Nucleic acids encoding the human ALEX1 protein

ABSTRACT

The present invention provides a novel protein containing an armadillo repeat, a gene encoding this protein, and production and use thereof. The present inventors identified a gene named ALEX1 encoding a human-derived novel armadillo repeat-containing protein. It was clarified that ALEX1 interacts with several proteins including insulin-degrading enzyme, presenilin-1, and JNK interacting protein 1. This gene shows significantly decreased expression in cancer cells. The protein ALEX1 and the gene encoding this protein are usable as tools in testing for diseases such as cancer and Alzheimer&#39;s disease and developing pharmaceutical agents.

TECHNICAL FIELD

This invention relates to a novel protein comprising a repetitivesequence called armadillo repeat, and a gene encoding this protein. Inaddition, this invention relates to the use of the obtained cDNA andproteins encoded by the cDNA in diagnosis of human cancer or Alzheimer'sdisease pathology.

BACKGROUND ART

Members of the armadillo (Arm) family of proteins are homologous to theproduct of the armadillo gene of Drosophila and have been implicated ina variety of important cell functions. The common characteristic of allarmadillo-related proteins is a series of imperfect 42-amino acidrepeats (Arm motifs) (Peifer, M. et al. (1994) Cell 76, 789-791). Themotif was first described in 1989 in the armadillo locus in Drosophila(Riggleman, B. et al. (1989) Genes Dev. 3, 96-113). Since then, a numberof genes encoding proteins containing homologous motifs have been clonedand sequenced. These include: human α-, β-, γ-, δ-catenins; the tumorsuppressor adenoma polyposis coli (APC); p120, the substrate of srcprotein kinases; importin involved in the nuclear import of proteins;and smgGDS involved in the guanine nucleotide conversion of lowmolecular weight G-proteins such as ras (Hatzfeld, M. (1999) Int. Rev.Cytol. 186, 179-224). Arm motifs are found throughout evolution and areconserved even between distant species like yeast and human. Arm repeatsin proteins usually occur in tandem and, so far, no proteins with lessthan six repeats have been described. It is known that armadillo-relatedproteins interact with numerous different proteins through their Armdomains (Ozawa, M. et al. (1995) J. Biochem. (Tokyo) 118, 1077-1082;Rubinfeld, B. et al. (1995) J. Biol. Chem. 270, 5549-5555; Troyanovsky,R. B. et al. (1996) J. Cell Sci. 109, 3069-3078; Murayama, M. et al.(1998) FEBS Lett. 433, 73-77).

Recent studies have implicated Arm proteins in Alzheimer's disease.Human Arm-proteins β-, γ-, δ-catenins and p0071 have been found tointeract with presenilin-1 (PS1) protein (Zhou, J. et al. (1997)NeuroReport 8, 2085-2090; Zhang, Z. et al. (1998) Nature 395, 698-702;Stahl, B. et al. (1999) J. Biol. Chem. 274, 9141-9148). Mutations of thePS1 and presenilin-2 (PS2) genes are responsible for the majority ofcases of early-onset familiar Alzheimer's disease (Price, D. L., andSisodia, S. S. (1998) Annu. Rev. Neurosci. 21, 479-505). PS1 and PS2mutations increase the levels of the β-amyloid peptide 1-42 deposited atthe core of amyloid plaques suggesting that presenilins are involved inthe processing of amyloid precursor proteins (APP). The significance ofinteraction of catenins with presenilins is unclear, although it appearsthat wild-type PS1 can stabilize β-catenin, whereas PS1 mutants showloss of the stabilizing function.

As mentioned above, Arm proteins serve different functions in cells. TheDrosophila armadillo gene was first identified as a component of thewingless signaling cascade (Wieschaus, E., and Riggleman, R. (1987) Cell49, 177-184). Similarly, the vertebrate equivalent of armadillo,β-catenin, is known as a critical component of the Wnt/Wingless growthfactor signaling pathway that governs cell fate choice in earlyembryogenesis. The mechanism of how β-catenin transduces theWnt/wingless signal has been elucidated by the discovery that β-cateninforms a complex with members of the TCF/LEF-1 family of transcriptionfactors that enters the nucleus (Behrens, J. et al. (1996) Nature 382,638-642; Molenaar, M. et al. (1996) Cell 86, 391-399). TCFs are poortranscriptional activators, but complexes of TCF/LEF-1 and β-catenin actas strong transcriptional activators.

In addition to its role in signaling functions, β-catenin has anessential role in the morphogenesis of solid tissues and the subsequentmaintenance of tissue integrity. β-catenin binds to the highly conservedcytoplasmic domain of cadherins and to α-catenin, which binds to actin.The cadherin-catenin complex is a target of regulatory signals thatgovern cellular adhesiveness and mobility (Kinch, M. S. et al. (1995) J.Cell. Biol. 130, 461-471).

In mammalian cells, β-catenin interacts with the tumor suppressor geneproduct APC (Su, L. K. et al. (1993) Science 262, 1734-1737). Mutationsin APC gene are associated with familial and sporadic colorectal cancer(Kinzler, K. W., and Vogelstein, B. (1996) Cell 87, 159-170). APC isthought to function to decrease β-catenin stability since APC mutantproteins lacking β-catenin binding site display elevated levels ofcytosolic β-catenin and constitutive transcriptional activation of theβ-catenin/TCF complex.

Thus, Arm proteins are involved in the maintenance of tissue structuresas well as intracellular signaling functions. They play a central rolein tumorigenesis and are implicated in Alzheimer's disease. Therefore,it is thought that newly discovered members of the Arm family ofproteins might be involved in these pathologies and, thus, representuseful targets for the development of therapeutics.

Recent studies from several laboratories demonstrated thatinsulin-degrading enzyme (IDE) is the main amyloid β peptide-degradingenzyme at neutral pH in rat and human nervous tissues (Kurochkin, I. V.,and Goto, S. (1994) FEBS Lett. 345, 33-37; McDermott, J. R., and Gibson,A. M. (1997) Neurochem. Res. 22, 49-56; Qiu, W. Q. et al. (1998) J.Biol. Chem. 273, 32730-32738). Since most of IDE is localized to thecytoplasm, it is unclear how the protease could gain access to amyloid βpeptide generated in a secretory organelle from an amyloid precursorprotein (APP).

DISCLOSURE OF THE INVENTION

An objective of this invention is to provide a novel protein comprisinga repetitive sequence called armadillo repeat, and a gene encoding thisprotein. Another objective of this invention is to provide the use ofthe obtained cDNA and proteins encoded by the cDNA in diagnosis of humancancer or Alzheimer's disease pathology and development ofpharmaceutical agents.

The present inventors hypothesized that IDE might get into contact withamyloid β peptide if adapter proteins exist linking the protease tocompartments where the peptide is generated. The inventors also thoughtthat a defect in function of putative adapter protein might beresponsible for accumulation of amyloid β peptide observed inAlzheimer's disease. Based on this hypothesis, the inventors used theyeast two-hybrid screen as an approach to find IDE potential proteinpartners that could fulfill the above-mentioned role. This led theinventors to the cloning of novel cDNA for a novel armadillo (Arm)repeat protein named ALEX1 (Arm protein Lost in Epithelial cancers onchromosome X-1), which has 453 amino acids.

In vitro experiments suggest a direct interaction between IDE and ALEX1protein. The protein predicted from its nucleotide sequence contains tworegions called armadillo repeats that are thought to be involved in theinteraction with other proteins. Homology search using the NCBI databaseshowed that ALEX1 shares significant homology with uncharacterizedKIAA0512, KIAA0443, and THC257925 corresponding to an open reading frameconsisting of several ESTs. The genes encoding ORF KIAA0512 and ORFbased on THC257925 are named as ALEX2 and ALEX3, correspondingly.Comparative amino acid sequence analysis allowed the inventors toconclude that ALEX1, ALEX2, ALEX3, and, probably, KIAA0443 constitute aseparate previously unidentified family of proteins. The genes for allfour members of the family are located at the same region on chromosomeX suggesting that these four ALEX genes evolved as a result ofamplification and divergence of a common ancestral gene related toclassical members of Arm repeat family of proteins.

Although overall homology of ALEX1 protein to other members of theArm-repeat family of proteins is insignificant, the region that spansArm repeats in ALEX1 is highly homologous to corresponding parts ofimportin-α and yeast Vac8p and to a less extent to segments inarmadillo, β-catenin, and plakoglobin. Importin-α (α-karyopherin) whichis known as a nuclear localization signal sequence receptor, is involvedin nuclear transport of proteins. It is known that yeast's Vac8p isinvolved in vacuole inheritance from mother cells to daughter cells(Wang, Y.-X. et al. (1998) J. Cell Biol. 140, 1063-1074). It may also bean essential component of the cytoplasm-to-vacuole targeting pathway asVac8p mutant yeast cells are defective in targeting of aminopeptidase Ifrom the cytoplasm to the vacuole. Interestingly, Vac8p protein isusually myristoylated in vivo (Wang, Y.-X. et al. (1998) J. Cell Biol.140, 1063-1074). Likewise, ALEX1 contains four putative myristoylationsites close to the N-terminus. ALEX1 protein may be involved intargeting cytosolic proteins into membrane-enclosed cell compartments ina manner similar to Vac8p.

Recent studies established that several members of Arm repeat familyincluding p0071, β-, γ-, and δ-catenins bind to presenilin-1 (PS1).Alzheimer's disease-linked PS1 mutations result in increased productionof the longer form of amyloid β peptide. New findings suggested that PS1regulates γ-secretase cleavage of APP or is itself a γ-secretase (DeStrooper, B. et al. (1998) Nature 391, 387-390; Wolfe, M. S. et al.(1999) Nature 398, 513-517). In this invention, the present inventorsdemonstrate that IDE interacts with ALEX1 in vitro. Further, it is shownthat ALEX1 can interact with PS1. Thus, ALEX1 protein could serve as anadapter protein for IDE to bring the protease into close proximity toPS1 and, therefore, to the site of amyloid β peptide production.Interestingly, PS1 mutations, which cause Alzheimer's disease, do notabolish its binding to Arm repeat proteins (Zhang, Z. et al. (1998)Nature 395, 698-702). It is considered that if ALEX1 forms a complexwith PS1, then mutations in ALEX1 or its reduced expression may disruptor weaken the complex formation to contribute to the pathology. In thisconnection, recent full genome scan for a causative gene of late onsetAlzheimer's disease identified amongst several candidate gene loci, asusceptibility locus in the region of X-chromosome (Kehoe et al. (1999)Hum. Mol. Genet. 8, 237-245) where ALEX1 is mapped, and anothersusceptibility locus in the long arm region of chromosome 10 (Bertram etal. (2000) Science, 290, 2302-2303, Ertekin-Taner et al. (2000) ibid,2303-2304, Myers et al. (2000) ibid, 2304-2305) where IDE is mapped. Byanalogy with functions of β-catenin and other members of Arm repeatfamily, it is suggested that ALEX1 may bind to presenilin-1 (PS1)through armadillo repeats in normal brain tissues, introduce IDE intothe site of amyloid β peptide production to contribute to thedegradation of amyloid β peptide, and regulate the accumulation ofamyloid β peptide.

Therefore, the ALEX1 protein can be a target molecule for preventing ortreating Alzheimer's disease, screening for drugs using the ALEX1 gene.Thus, ALEX1 protein enables the development of novel therapeutic agentsfor Alzheimer's disease, and in addition, by detecting the expression ofALEX, it enables the diagnosis and testing of Alzheimer's disease.

Expression studies demonstrated that ALEX1 is expressed in almost allhuman tissues, except leukocytes (FIG. 4). The highest expression wasrevealed in the heart, skeletal muscle, brain, ovary, and prostate. Itwas barely detectable in liver and thymus. ALEX1 was expressed in allregions of the human brain tested. The lack of ALEX1 expression only inperipheral blood leukocytes suggests that this protein might be involvedin signal transduction mediated by some sort of intercellular adhesionor establishment of cell polarity. Indeed, of all tissues examined, onlyleukocytes exist as single cells and only leukocytes lack any long-termestablished polarity and display highest motility. On the other hand,tissues with maximal ALEX1 expression, brain, heart, and skeletalmuscle, consist of cells with the most striking polarized organizationof any cell type in the body.

Mouse ortholog of ALEX1 is developmentally regulated with significantinduction between days of 7 and 11 of embryo development (FIG. 8). Thisperiod in the development is associated with early organogenesissuggesting that ALEX1 might be involved in the morphogenesis and/orspecification of embryonic patterning as another Arm repeat proteinβ-catenin, a component of Wnt-signaling pathway.

ALEX1 and ALEX2 showed remarkably similar expression patterns. LikeALEX1, ALEX2 protein contains an N-terminal transmembrane domain with amost likely targeting to the endoplasmic reticulum membrane. The moststriking finding is significantly reduced or undetectable expression ofALEX1 and ALEX2 in human tumors and tumor-derived cell lines (FIGS. 5,6, and 7). In this invention, the inventors demonstrated that ALEX1 andALEX2 transcripts are lost or reduced in cell lines derived from humancarcinomas, but not from neuroblastomas, glioblastomas, or sarcomas.Carcinomas, the cancers of epithelial tissues, represent about 70% ofall human tumors. The fact that ALEX1 and ALEX2 expression is impairedin carcinomas of multiple tissues suggests that these proteins aregeneral factors involved in regulation of normal cell growth. Verysimilar expression patterns in normal and cancer tissues indicate thatboth genes are under the control of the same transcription factors.

Expression of ALEX1 and ALEX2 mRNA is lost or significantly reduced inhuman carcinoma samples and in cell lines established from various humancarcinomas. These genes are however normally expressed in cell linesderived from other types of human tumors, i.e. sarcomas, neuroblastomas,and gliomas. Based on these findings, the present invention providesnovel methods for diagnosing epithelial tumors using, as an index, thepresence or absence of mutations in ALEX1 and ALEX2 genes, or reducedexpression thereof. Cancer may be prevented or treated by using drugsregulating the expression of ALEX1 gene, or the activity of ALEX1protein.

In order to gain insight into ALEX1's physiological role, the presentinventors employed the yeast two-hybrid screen to identify potentialALEX1-interacting proteins. Thereby, the inventors identified a group ofthree overlapping clones derived from cDNA for JNK interacting protein-1(JIP-1). JIP-1 is a recently cloned scaffold protein for the componentsof JNK signaling pathway: MLK3 (MAPKKK)->MKK7 (MAPKK)->JNK (MAPK). JIP-1facilitates signaling by the bound protein kinases (Whitmarsh, A. J. etal. (1998) Science 281, 1671-1674). MKK7 is a major activator for JNK inthe TNFα-stimulated pathway and in osmotically shocked cells. Theinventors utilized c-Jun-dependent reporter gene system to analyze invivo effect of ALEX1 on c-Jun-dependent transcriptional activation. As aresult, expression of ALEX1 markedly inhibited c-Jun-dependenttranscriptional activation in a dose-dependent manner. Studies withvarious JIP-1 deletion mutants established that the binding site forALEX1 on JIP-1 overlaps with the reported MKK7-binding domain, but notJNK and MLK3. Therefore, ALEX1 might negatively regulate JNK signalingcascade by competitively inhibiting the binding of MKK7 and the scaffoldprotein JIP-1. In this scenario, since ALEX1 inhibits the step of MKK7,the signal transduction from upstream component of the cascade, MLK3, todownstream effector, JNK, will be interrupted. This negative regulationdisappears in cancer cells where the expression of ALEX1 is reduced orlost, and thus, c-Jun as well as JNK may be activated, contributing totumorigenesis.

Furthermore, as a result of screening for proteins that interact withALEX1 using yeast two-hybrid screening that uses ALEX1 lacking thetransmembrane domain at the N-terminus (amino acid numbers 1 to 27) as abait, the present inventors found that p0071 (plakophilin-4) SART-1,MSP58, ATRX, CSA2 (RED protein), p68 (RNA helicase/ATPase) OS-9, ZNF189,KIAA1221, α-Actinin4, and ZIP kinase interact with the above-mentionedALEX1 protein lacking the N-terminal transmembrane domain.

By using these activities of the ALEX1 protein as indexes to screen forcompounds that regulate the activity of the ALEX1 protein, drugseffective for the prevention and treatment of cancer and Alzheimer'sdisease can be obtained.

That is, the ALEX1 protein of this invention and the gene encoding thisprotein are thought to be useful as indexes for testing diseases such ascancer and Alzheimer's disease, as tools to elucidate the pathologicalmechanism of these diseases, and furthermore, as targets for developingpharmaceutical agents against these diseases.

This invention relates to a novel “ALEX1” protein, a gene encoding thisprotein, and production and use thereof, and more specifically relatesto,

(1) a DNA encoding a protein having an armadillo repeat structure,wherein the DNA is selected from the group consisting of:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 3,

(b) a DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1 or 2,

(c) a DNA encoding a protein in which one or more amino acids of theamino acid sequence of SEQ ID NO: 3 has been substituted, deleted,inserted, and/or added, and

(d) a DNA that hybridizes under stringent conditions to a DNA comprisingthe nucleotide sequence of SEQ ID NO: 1 or 2,

(2) a DNA encoding a protein that binds to a protein selected from thegroup consisting of insulin-degrading enzyme (IDE), presenilin-1 (PS-1),p0071 (plakophilin-4), SART-1, MSP58, ATRX, CSA2 (RED protein), p68 (RNAhelicase/ATPase), OS-9, ZNF189, KIAA1221, α-Actinin4, and ZIP kinase,wherein the DNA is selected from the group consisting of:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 3,

(b) a DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1 or 2,

(c) a DNA encoding a protein in which one or more amino acids of theamino acid sequence of SEQ ID NO: 3 has been substituted, deleted,inserted, and/or added, and

(d) a DNA that hybridizes under stringent conditions to a DNA comprisingthe nucleotide sequence of SEQ ID NO: 1 or 2,

(3) a DNA encoding a protein that binds to JNK interacting protein 1(JIP-1), wherein the DNA is selected from the group consisting of:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 3,

(b) a DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1 or 2,

(c) a DNA encoding a protein in which one or more amino acids of theamino acid sequence of SEQ ID NO: 3 has been substituted, deleted,inserted, and/or added, and

(d) a DNA that hybridizes under stringent conditions to a DNA comprisingthe nucleotide sequence of SEQ ID NO: 1 or 2,

(4) the DNA of (3) that encodes a protein that inhibits c-Jun-dependenttranscription,

(5) a DNA encoding a partial peptide of a protein comprising the aminoacid sequence of SEQ ID NO: 3,

(6) a protein or peptide encoded by the DNA of any one of (1) to (5)

(7) a vector into which the DNA of any one of (1) to (5) has beeninserted,

(8) a host cell carrying the DNA of any one of (1) to (5) or the vectorof (7),

(9) a method of producing the protein or peptide of (6), the methodcomprising the steps of cultivating the host cell of (8) and collectingthe expressed protein from the host cell or culture supernatant thereof,

(10) an antibody that binds to the protein of (6),

(11) a polynucleotide comprising at least 15 nucleotides complementaryto a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 2, or tocomplementary strand thereof,

(12) a polynucleotide comprising at least 15 nucleotides complementaryto the DNA of any one of (1) to (4) or to complementary strand thereof,wherein the polynucleotide is used for testing Alzheimer's disease orcancer,

(13) a method of screening for a compound that binds to the protein of(6), the method comprising the steps of,

(a) contacting a test sample with the protein or partial peptidethereof,

(b) detecting binding activity between the test sample and the proteinor partial peptide thereof, and

(c) selecting a compound that binds to the protein or partial peptidethereof,

(14) a method of screening for a compound that regulates the expressionof the DNA of any one of (1) to (4), the method comprising the steps of,

(a) contacting a test sample with a cell that endogenously expresses theDNA,

(b) detecting the expression, and

(c) selecting a compound that promotes or inhibits the expressioncompared to when the cell is not contacted with the test sample,

(15) a method of screening for a compound that regulates the expressionof the DNA of any one of (1) to (4), the method comprising the steps of,

(a) contacting a test sample with a cell into which a vector having areporter gene operably linked downstream of an endogenous transcriptionregulatory sequence of the DNA of any one of (1) to (4) has beenintroduced,

(b) detecting expression of the reporter gene within the cell, and

(c) selecting a compound that promotes or inhibits expression of thereporter gene compared to when the cell is not contacted with the testsample,

(16) a method of screening for a compound that regulates binding betweena protein encoded by the DNA of (2) and a protein selected from thegroup consisting of insulin-degrading enzyme (IDE), presenilin-1 (PS-1),p0071 (plakophilin-4), SART-1, MSP58, ATRX, CSA2 (RED protein), p68 (RNAhelicase/ATPase), OS-9, ZNF189, KIAA1221, α-Actinin4, and ZIP kinase,wherein the method comprises the steps of,

(a) contacting a protein encoded by the DNA of (2) with a proteinselected from the group consisting of insulin-degrading enzyme (IDE),presenilin-1 (PS-1), p0071 (plakophilin-4), SART-1, MSP58, ATRX, CSA2(RED protein), p68 (RNA helicase/ATPase), OS-9, ZNF189, KIAA1221,α-Actinin4, and ZIP kinase, in the presence of a test sample,

(b) detecting binding of these proteins, and

(c) selecting a compound that promotes or inhibits binding of theseproteins compared to when detected in the absence of the test sample,

(17) a method of screening for a compound that regulates the bindingbetween a protein encoded by a DNA of (3) and JIP-1 protein, wherein themethod comprises the steps of,

(a) contacting the protein encoded by the DNA of (3) with JIP-1 proteinin the presence of a test sample,

(b) detecting the binding of these proteins, and

(c) selecting a compound that promotes or inhibits the binding of theseproteins compared to when the detection is performed in the absence ofthe test sample,

(18) a method of screening for a compound that regulates the activity ofa protein encoded by a DNA of (3), wherein the method comprises thesteps of,

(a) contacting a test sample with a cell expressing the protein encodedby the DNA of (3),

(b) detecting MKK7/JNK-mediated signal transduction in the cell, and

(c) selecting a compound that promotes or inhibits the signaltransduction,

(19) the method of (18), wherein the MKK7/JNK-mediated signaltransduction is detected using c-Jun-dependent transcription as an indexin step (b),

(20) a compound that can be isolated by the method of any one of (13) to(19)

(21) a pharmaceutical composition comprising the compound of (20) as anactive ingredient,

(22) the pharmaceutical composition of (21) for preventing or treatingAlzheimer's disease or cancer,

(23) a method for testing Alzheimer's disease or cancer, the methodcomprising the step of detecting the expression level of DNA encodingthe protein of (6) in a patient-derived test sample,

(24) the method of (23), wherein the expression level of the DNA isdetected by a method comprising the steps of,

(a) contacting the polynucleotide of (12) with a patient-derived RNAsample, and

(b) detecting the binding of the polynucleotide to the RNA sample,

(25) the method of (24), wherein the expression level of the DNA isdetected by a method comprising the steps of,

(a) synthesizing cDNA from a patient-derived RNA sample,

(b) performing polymerase chain reaction using the synthetic cDNA astemplate and the polynucleotide of (12) as a primer, and

(c) detecting DNA amplified by polymerase chain reaction,

(26) a method for testing Alzheimer's disease or cancer, wherein themethod comprises the step of detecting the level of the protein of (6)present in a patient-derived test sample,

(27) the method of (26), wherein the level of protein present isdetected by a method comprising the steps of,

(a) contacting the antibody of (10) with a patient-derived proteinsample, and

(b) detecting the binding of the antibody to the protein sample,

(28) a method for testing Alzheimer's disease or cancer, the methodcomprising detecting a mutation in DNA encoding the protein of (6)

(29) the method for testing Alzheimer's disease or cancer of (28), themethod comprising the steps of determining the nucleotide sequence ofDNA encoding the protein of (6) and comparing it with the sequence of ahealthy person,

(30) the method of (29), wherein the mutation is detected by a methodcomprising the steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying the patient-derived DNA using the polynucleotide of (12)as a primer,

(c) cleaving the amplified DNA,

(d) separating the DNA fragments according to their size,

(e) hybridizing the polynucleotide of (12) that has a detectable labelas a probe to the separated DNA fragments, and

(f) comparing the detected DNA fragment size to a control from a healthyperson,

(31) the method of (29), wherein the mutation is detected by a methodcomprising the steps of,

(a) preparing an RNA sample from a patient,

(b) separating the prepared RNA according to size,

(c) hybridizing the polynucleotide of (12) that has a detectable labelas a probe to the separated RNA, and

(d) comparing the detected RNA size to a control from a healthy person,

(32) the method of (29), wherein the mutation is detected by a methodcomprising the steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying the patient-derived DNA using the polynucleotide of (12)as a primer,

(c) dissociating the amplified DNA into single stranded DNA,

(d) separating the dissociated single stranded DNA on a non-denaturinggel, and

(e) comparing the mobility of the separated single stranded DNA on thegel with that of a control from a healthy person,

(33) the method of (29), wherein the mutation is detected by a methodcomprising the steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying the patient-derived DNA using the polynucleotide of (12)as a primer,

(c) separating the amplified DNA on a gel in which DNA denaturantconcentration gradually rises, and

(d) comparing the mobility of the separated DNA on the gel with that ofa control from a healthy person,

(34) a method for testing Alzheimer's disease or cancer, the methodcomprising detecting a mutation in the protein of (6),

(35) the method of (34), wherein the mutation is detected using thebinding activity with a protein selected from the group consisting ofIDE, PS-1, JIP-1, p0071 (plakophilin-4), SART-1, MSP58, ATRX, CSA2 (REDprotein), p68 (RNA helicase/ATPase), OS-9, ZNF189, KIAA1221, α-Actinin4,and ZIP kinase as an index,(36) a testing reagent for Alzheimer's disease or cancer, comprising theantibody of (10), and(37) a testing reagent for Alzheimer's disease or cancer, comprising thepolynucleotide of (12).

This invention relates to a gene “ALEX1” encoding a novel armadillorepeat-containing protein. The nucleotide sequences of human-derivedALEX1 cDNA are shown in SEQ ID NOS: 1 and 2, and the amino acid sequenceof the protein encoded by these cDNA is shown in SEQ ID NO: 3. As SEQ IDNOS: 1 and 2 indicate, human ALEX1 cDNA has an ORF encoding a proteincomprising 453 amino acids. Since human ALEX1 protein of this inventionbinds to IDE, and interacts with presenilin-1, it is thought to beinvolved with the onset of Alzheimer's disease.

In addition, by Northern blotting and RT-PCR analysis, expression ofhuman ALEX1 gene was observed in various tissues, and was especiallyhigh in the ovary, heart, testis, prostate, brain, spleen, skeletalmuscle, and colon, and in contrast, expression was weak in the liver andthymus. Also, mRNA expression in the peripheral leukocytes was at orbelow the detection limit. Also, as a result of analyzing expression ofALEX1 using cancer cells and cancer tissues, it was elucidated thatexpression of ALEX1 is significantly reduced in epithelial cancer.Therefore, by testing the expression of ALEX1 of this invention, it ispossible to test for cancer.

Furthermore, this invention includes proteins functionally equivalent tohuman “ALEX1” protein (SEQ ID NO: 3). Such proteins include, forexample, mutants of human “ALEX1” protein and homologues of organismsother than human. Such proteins include for example, proteins having anarmadillo repeat structure. Whether a protein has an armadillo repeatstructure can be determined through analysis by Pfam algorithm (Bateman,A. et al., Nucleic Acids Research 27:260-262 (1999)) using the obtainedamino acid sequence as a query.

Analysis by Pfam can be performed for example, by using the system atSanger centre (Sanger Institute, Cambridge, UK). The cutoff value forE-value is set at 20. If the E-value is 20 or less (at higher precision,15 or less), the amino acid sequence of the searched protein isdetermined to have an armadillo repeat structure. For example, for ALEX1(SEQ ID NO: 3), the first arm repeat (amino acid numbers 193 to 235) isdetected at E-value=11 and the second arm repeat is detected atE-value=2.1. Similarly, for KIAA0152 (ALEX2) and ALEX3, one arm repeatis detected at amino acid numbers 416 to 457 (E-value=0.56) and at aminoacid numbers 151 to 192 (E-value=9.3), respectively. Also, in ananalysis performed on ProfileScan Server, one arm repeat is detected ata normalized score of 8.802 at amino acid numbers 247 to 284 of ALEX1,and the obtained result is a significant match.

In addition, proteins functionally equivalent to the human “ALEX1”protein (SEQ ID NO: 3), include a protein that has an activity to bindto insulin-degrading enzyme (IDE) protein (Kurochkin, I. V., and Goto,S. (1994) FEBS Lett. 345, 33-37; McDermott, J. R., and Gibson, A. M.(1997) Neurochem. Res. 22, 49-56; Qiu, W. Q. et al. (1998) J. Biol.Chem. 273, 32730-32738).

The binding activity of a certain protein with an IDE protein can bedetected with high sensitivity, for example, by immunoprecipitation ofthe IDE protein by an anti-IDE antibody, or precipitation of IDE towhich a specific tag has been added by an anti-tag antibody, and whendetecting the co-precipitating protein, a tag is added to the targetprotein, and detection can be made by an antibody against this tag, orby using a specific antibody against the target protein. Other methodsfor detecting the binding activity with the IDE protein includepull-down method, which does not use antibodies and analyzes proteinsadsorbed by mixing IDE bound to beads with a cell extract solution.Alternatively, a method that directly observes the binding by BIAcore,and furthermore, a method that functionally observes the binding in aliving cell by Two-Hybrid method can also be used.

For the IDE protein used above, a wild type protein may be used, but anIDE protein to which mutations have been introduced may also be used.For example, H108Q mutation shown in Examples maintains substratebinding activity of IDE protein while destroying protease activity. Byintroducing this mutation, when binding assay is performed within yeastcells and such, cytotoxic activity of the IDE protein toward host cellscan be removed, and when a protein bound to IDE is the substrate of IDE,bond breakage upon dissociation can be avoided. Therefore, for a morestable detection of binding, it is considered preferable to use H108Q.Also, as long as proteins binding to IDE can be detected stably, othermutant IDE proteins may be used.

In addition, as proteins functionally equivalent to human “ALEX1”protein (SEQ ID NO: 3), proteins having activity to bind to presenilin-1protein (Kimberly, W T et al. J. Biol. Chem. 2000, 275 (5) 3173-8;Selkoe, D J. Trends Cell Biol. 1998, 8, 447-453) are included.Furthermore, proteins having activity to bind to JNK interacting protein1 (JIP-1) (Whitmarsh, A. J. et al. (1998) Science 281, 1671-1674) areincluded. Whether or not there is a binding activity with the PS-1protein or the JIP-1 protein can be determined by immunoprecipitation,pull-down method, BIAcore, Two-Hybrid method, and such, similarly to thedetection of the binding activity with the IDE protein mentioned above.

The protein of this invention having activity to bind to JIP-1preferably has the activity to inhibit c-Jun-dependent transcription.Whether or not there is an activity to inhibit c-Jun-dependenttranscription or not can be determined for example, as indicated inExamples, by using a c-Jun-dependent luciferase reporter construct, andby transfecting a vector expressing the test protein of interest withMEKK1 expression vector for example, and whether the activation ofc-Jun-dependent transcription by MEKK1 is inhibited by the test proteinor not can be determined using expression of the reporter gene as anindex.

Furthermore, proteins functionally equivalent to human “ALEX1” protein(SEQ ID NO: 3) include proteins having activity to bind to the proteinsselected from the group consisting of p0071 (plakophilin-4), SART-1,MSP58, ATRX, CSA2 (RED protein), p68 (RNA helicase/ATPase), OS-9,ZNF189, KIAA1221, α-Actinin4, and ZIP kinase. Whether or not there is abinding activity with these proteins can be determined byimmunoprecipitation, pull-down method, BIAcore, Two-Hybrid method, andsuch, similarly to the detection of binding activity with the IDEprotein mentioned above. Examples of these proteins are, specifically,proteins encoded by the genes shown below.

p0071 (plakophilin-4): Arm repeat-containing presenilin-binding protein

-   -   GenBank Ac. No. X81889; H. sapiens mRNA for p0071 protein,        GenBank Ac. No. NM_(—)003628; Homo sapiens plakophilin 4 (PKP4),        mRNA        SART-1: human squamous cell carcinoma antigen, expressed in        growing cells only    -   GenBank Ac. No. AB006198; Homo sapiens mRNA for SART-1, GenBank        Ac. No. Y14314; Homo sapiens mRNA for IgE autoantigen        MSP58: nucleoprotein, interacting to the growth-related        nucleoprotein p120, expressed in growing cells only    -   GenBank Ac. No. AF015308; Homo sapiens nucleolar protein (MSP58)        mRNA, GenBank Ac. No. AF068007; Homo sapiens cell        cycle-regulated factor p78 mRNA        ATRX: murine colon adenocarcinoma antigen, cell        cycle-dependently phosphorylated, a helicase/ATPase member of        the SNF2 family    -   GenBank Ac. No. U72938; Homo sapiens putative DNA dependent        ATPase and helicase (ATRX) mRNA, alternatively spliced product        3, GenBank Ac. No. NM_(—)000489; Homo sapiens alpha        thalassemia/mental retardation syndrome X-linked (RAD54 (S.        cerevisiae) homolog) (ATRX), mRNA        CSA2 (RED protein): chondrosarcoma-associated protein,        distributed in nuclei as dots, transcription-related function?    -   GenBank Ac. No. AF182645; Homo sapiens chondrosarcoma-associated        protein 2 (CSA2) mRNA        p68: RNA helicase/ATPase    -   GenBank Ac. No. X52104; Human mRNA for p68 protein, GenBank Ac.        No. NM_(—)004396; Homo sapiens DEAD/H (Asp-Glu-Ala-Asp/His) box        polypeptide 5 (RNA helicase, 68 kD) (DDX5), mRNA        OS-9: amplified in human sarcoma, containing nuclear        localization signal    -   GenBank Ac. No. NM_(—)006812; Homo sapiens amplified in        osteosarcoma (OS-9), mRNA        ZNF189: C₂H₂ Zinc Finger protein    -   GenBank Ac. No. AF025770; Homo sapiens C2H2 zinc finger protein        (ZNF189) mRNA        KIAA1221: C₂H₂ Zinc Finger protein    -   GenBank Ac. No. AB033047; Homo sapiens mRNA for KIAA1221        protein, partial cds        α-Actinin4: involved in actin polymerization, cell motility, and        cancer infiltration    -   GenBank Ac. No. NM_(—)004924; Homo sapiens actinin, alpha 4        (ACTN4) mRNA        ZIP kinase    -   GenBank Ac. No. AB022341; Homo sapiens mRNA for ZIP kinase

One method well known to those skilled in the art for preparingfunctionally equivalent proteins is to introduce mutations intoproteins. For example, one skilled in the art can prepare proteinsfunctionally equivalent to the human “ALEX1” protein by introducingappropriate mutations into the amino acid sequence of the protein (SEQID NO: 3), by using site-specific mutagenesis (Hashimoto-Gotoh, T. etal. (1995) Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) MethodsEnzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res. 12,9441-9456; Kramer W, and Fritz H J (1987) Methods Enzymol. 154, 350-367;Kunkel, T A (1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988)Methods Enzymol. 85, 2763-2766), and such. Mutation of amino acids mayoccur in nature, too. Furthermore, the proteins of the present inventioninclude a protein comprising an amino acid sequence of the “ALEX1”protein (SEQ ID NO: 3) in which one or more amino acids have beenmutated, which is functionally equivalent to the ALEX1 protein. In sucha mutant protein, the number of amino acids mutated are considered to beusually 100 residues or less, preferably 50 residues or less, morepreferably 30 residues or less, even more preferably 10 residues or less(e.g. 5 residues or less).

It is preferable to mutate an amino acid residue into one that allowsthe properties of the amino acid side-chain to be conserved. Examples ofproperties of amino acid side chains include: hydrophobic amino acids(A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q,G, H, K, S, T), and amino acids comprising the following side chains:aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containingside-chains (S, T, Y); sulfur atom-containing side-chains (C, M);carboxylic acid- and amide-containing side-chains (D, N, E, Q);base-containing side-chain (R, K, H); and aromatic-containingside-chains (H, F, Y, W) (The letters within parenthesis indicate theone-letter codes of amino acids). Proteins whose amino acids have beenconservatively substituted are included in the proteins of thisinvention.

It is well known that a protein having a deletion, addition, and/orsubstitution of one or more amino acid residues in the sequence of theprotein can retain the original biological activity (Mark D. F. et al.Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666 (1984); Zoller M. J. andSmith M. Nucleic Acids Res. 10:6487-6500 (1982); Wang A. et al. Science224:1431-1433 (1984); Dalbadie-McFarland G. et al. Proc. Natl. Acad.Sci. U.S.A. 79:6409-6413 (1982)).

A protein in which amino acid residues have been added to the amino acidsequence of the human “ALEX1” protein includes a fusion proteincomprising the human “ALEX1” protein. The present invention includes afusion protein in which the human “ALEX1” protein and one or more otherproteins or peptides are fused. Methods well known in the art may beused to generate a fusion protein of the present invention. For example,DNA encoding the human “ALEX1” protein (SEQ ID NO: 3) and DNA encodinganother protein or peptide are linked in frame and introduced into anexpression vector. The fusion protein is then expressed in a host cell.The protein or peptide fused to a protein of the present invention isnot limited to any specific protein or peptide.

Known peptides, for example, FLAG (Hopp, T. P. et al., Biotechnology(1988) 6, 1204-1210), 6×His containing six His (histidine) residues,10×His, Influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, andsuch, can be used as peptides that are fused to a protein of the presentinvention. Examples of proteins that are fused to a protein of theinvention are, GST (glutathione-S-transferase), Influenza agglutinin(HA), immunoglobulin constant region, β-galactosidase, MBP(maltose-binding protein), and such. Fusion proteins can be prepared byfusing commercially available DNA encoding these peptides or proteinswith DNA encoding a protein of the present invention and expressing thefused DNA prepared.

An alternative method known to those skilled in the art for preparingfunctionally equivalent proteins is, for example, the method utilizingthe hybridization technique (Sambrook, J. et al., Molecular Cloning 2nded. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Generally, oneskilled in the art can isolate DNA highly homologous to the whole orpart of a DNA sequence encoding the human “ALEX1” protein (SEQ ID NO: 1or 2), and then isolate a protein functionally equivalent to the human“ALEX1” protein from those DNA isolated. The present invention includesproteins encoded by DNA that hybridize with the whole or part of DNAencoding the human “ALEX1” protein, in which the proteins arefunctionally equivalent to the human “ALEX1” protein. These proteinsinclude homologues of nonhuman mammal (e.g. proteins encoded by genes ofmonkeys, mice, rats, rabbits, and cattle). Also, since the ALEX genesform a family, it is possible to isolate other ALEX family genes usingDNA encoding human “ALEX1” protein as probes and primers. For example, aprobe or a primer can be constructed based on the amino acid sequence ofALEX1, particularly the 226 amino acid-long sequence from amino acidnumber 200 to the C-terminal end containing an Arm repeat, or a portionthereof. Thereafter, hybridization and amplification by PCR may beperformed under an appropriate stringency. When isolating animal-derivedcDNA highly homologous to DNA encoding human “ALEX1” protein, manytissues and cells, including ovary, heart, testis, prostate, brain,spleen, skeletal muscle, and colon, expected to express the protein ofthis invention may be used, but the source is not limited thereto.

Hybridization conditions for isolating DNA encoding a proteinfunctionally equivalent to the human “ALEX1” protein may beappropriately selected by a person skilled in the art. A stringenthybridization condition is, for example, washing in 42° C., 2×SSC, 0.1%SDS, after hybridization, and preferably, in 50° C., 2×SSC, 0.1% SDS.More preferably, for example, washing is conducted in 65° C., 2×SSC,0.1% SDS. Under these conditions, the higher the temperature, the higherthe homology of the obtained DNA will be. However, several factors, suchas temperature and salt concentration, can influence the stringency ofhybridization and one skilled in the art can suitably select the factorsto accomplish a similar stringency.

In place of hybridization, a gene amplification method using primerssynthesized based on the sequence information of the DNA (SEQ ID NO: 1or 2) encoding the human “ALEX1” protein, for example, the polymerasechain reaction (PCR) method, can be utilized.

A protein functionally equivalent to the human “ALEX1” protein encodedby the DNA isolated through the above hybridization technique or geneamplification techniques normally has a high homology to the amino acidsequence of the human “ALEX1” protein (SEQ ID NO: 3). The proteins ofthe present invention also include proteins that are functionallyequivalent to the human “ALEX1” protein and are highly homologous to theamino acid sequence shown in SEQ ID NO: 3. “Highly homologous” refersto, normally an identity of at least 60% or higher, preferably 75% orhigher, more preferably 90% or higher, and even more preferably 95% orhigher, at the amino acid level. The homology of a protein can bedetermined by following the algorithm in “Wilbur, W. J. and Lipman, D.J. (1983) Proc. Natl. Acad. Sci. USA 80:726-730”.

The proteins of the present invention may have variations in the aminoacid sequence, molecular weight, isoelectric point, the presence orabsence of sugar chains, form, and so on, depending on the cell or hostused to produce it or the purification method utilized (describedbelow). Nevertheless, as long as the obtained protein has a functionequivalent to the human “ALEX1” protein, it is within the scope of thepresent invention.

The proteins of the present invention can be prepared as recombinantproteins or naturally occurring proteins, using methods commonly knownin the art. When the protein is a recombinant protein, it may beproduced by inserting DNA (for example, DNA having the nucleotidesequence of SEQ ID NO: 1 or 2) encoding a protein of the presentinvention into an appropriate expression vector, collecting thetransformant obtained by introducing the vector into an appropriate hostcell, obtaining an extract, and then purifying and preparing the proteinusing ion exchange, reverse phase, gel filtration, or affinitychromatography. Affinity chromatography may be done using a column inwhich an antibody against a protein of the present invention isimmobilized. A combination of such columns may also be used.

Alternatively, when a protein of the invention is expressed in hostcells (e.g., animal cells or E. coli) as a fusion protein withglutathione S transferase protein, or a recombinant protein withmultiple histidine residues, the expressed recombinant protein can bepurified using a glutathione column or nickel column. After the fusionprotein is purified, if necessary, regions of the fusion protein (apartfrom the desired protein) can be digested and removed with thrombin,factor Xa, etc.

The native protein of the invention can be isolated by methods wellknown in the art, for example, purifying an extract of tissues or cellsthat express a protein of the invention with an affinity column to whichan antibody binding to a protein of the present invention describedbelow is bound. The antibody may be a polyclonal or monoclonal antibody.

The present invention also includes partial peptides of the proteins ofthe present invention. The partial peptides of the present inventioncomprise at least 7 or more amino acids, preferably 8 or more aminoacids, more preferably 9 or more amino acids. The partial peptides canbe used, for example, for generating antibodies against a protein of thepresent invention, screening of compounds binding to a protein of thepresent invention, or screening of stimulators or inhibitors of aprotein of the present invention. Additionally, they may be antagonistsor competitive inhibitors of the proteins of the present invention. Thepartial peptides of the proteins of the present invention include thosethat include, for example, functional domains of the human “ALEX1”protein (SEQ ID NO: 3). Such functional domains include, for example,the Arm domain. A partial peptide containing one or more Arm domains(Arm1 and/or Arm2) (see FIG. 2) is included in the partial peptides ofthe present invention. In addition, a partial protein of “ALEX1” inwhich the transmembrane domain has been deleted (e.g. a protein without1^(st) to 27^(th) amino acids) may also be included. The partialpeptides of the present invention can be produced by genetic engineeringmethods, known peptide synthesis methods, or by cutting the proteins ofthe present invention by appropriate peptidases. Synthesis of thepeptides may be conducted according to, for example, the solid phasesynthesis method, or the liquid phase synthesis method.

In addition to being utilized in the above-described in vivo or in vitroproduction of a protein of the present invention, DNA encoding a proteinof the present invention may also be applied, for example, in the genetherapy of diseases caused by an aberration in a gene encoding a proteinof the present invention or diseases treatable by a protein of thepresent invention. Any type of DNA, such as cDNA synthesized from mRNA,genomic DNA, or synthetic DNA can be used so long as the DNA encodes aprotein of the present invention. Also as long as they can encode aprotein of the present invention, DNA comprising arbitrary sequencesbased on the degeneracy of the genetic code are also included.

The DNA of the present invention can be prepared by using methods knownin the art. For example, a cDNA library can be constructed from cellsexpressing a protein of the present invention and hybridization can beconducted using a part of the DNA sequence of the present invention (forexample, SEQ ID NO: 1 or 2) as a probe. The cDNA library may beprepared, for example, according to the method described by Sambrook J.et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)),or instead, commercially available cDNA libraries may be used.Alternatively, DNA of the present invention can be obtained by preparingRNA from cells expressing a protein of the present invention,synthesizing cDNA by using a reverse transcriptase, synthesizingoligo-DNA based on a DNA sequence of the present invention (for example,SEQ ID NO: 1 or 2), and amplifying the cDNA encoding a protein of thepresent invention by PCR using the oligo-DNA as primers.

The nucleotide sequence of the obtained cDNA is determined to find anopen reading frame, and thereby, the amino acid sequence of a protein ofthe invention can be obtained. The cDNA obtained may also be used as aprobe for screening a genomic library to isolate genomic DNA. Similarly,endogenous transcriptional regulators of genes encoding proteins of thisinvention can be obtained.

More specifically, mRNA may first be isolated from a cell, tissue, ororgan in which a protein of the invention is expressed (e.g. tissuessuch as ovary, heart, testis, prostate, brain, spleen, skeletal muscle,and colon). Known methods can be used to isolate mRNA; for instance,total RNA is prepared by guanidine ultracentrifugation (Chirgwin J. M.et al. Biochemistry 18:5294-5299 (1979)) or AGPC method (Chomczynski P.and Sacchi N. Anal. Biochem. 162:156-159 (1987)), and mRNA is purifiedfrom total RNA using an mRNA Purification Kit Pharmacia) and such.Alternatively, mRNA may be directly prepared by QuickPrep mRNAPurification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a kit such as the AMVReverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, cDNA may be synthesized and amplified following the5′-RACE method or 3′-RACE method (Frohman M. A. et al. Proc. Natl. Acad.Sci. U.S.A. 85:8998-9002 (1988); Belyavsky A. et al. Nucleic Acids Res.17:2919-2932 (1989)) that uses primers and such described herein, the5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction(PCR).

A desired DNA fragment is prepared from the PCR products and linked tovector DNA. The recombinant vector is used to transform E. coli andsuch, and the desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA can be verified byconventional methods, such as dideoxynucleotide chain termination.

DNA of the invention may be designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host used for expression (Grantham R. et al. Nucleic AcidsRes. 9:43-74 (1981)). The DNA of the present invention may be altered bya commercially available kit or a conventional method. For instance, theDNA may be altered by digestion with restriction enzymes, insertion of asynthetic oligonucleotide or an appropriate DNA fragment, addition of alinker, or insertion of the initiation codon (ATG) and/or the stop codon(TAA, TGA, or TAG), etc.

Specifically, the DNA of the present invention include DNA having thefollowing nucleotide sequences: from A at position 372 to C at position1730 of SEQ ID NO: 1, and from A at position 364 to C at position 1722of SEQ ID NO: 2.

Furthermore, the DNA of the present invention include DNA capable ofhybridizing with DNA having the nucleotide sequence of SEQ ID NO: 1 or2, and encoding a protein functionally equivalent to a protein of theinvention described above. Hybridization conditions may be appropriatelychosen by one skilled in the art. Specifically, conditions describedabove may be used. Under the conditions, DNA having higher homologiescan be obtained by increasing temperature. The above hybridizing DNA ispreferably natural DNA, for example, cDNA or chromosomal DNA.

The present invention also provides a vector into which DNA of thepresent invention is inserted. The vectors of the present invention areuseful in maintaining the DNA of the present invention within the hostcell, or expressing a protein of the present invention.

When E. coli is used as the host cell, there is no limitation other thanthat the vector should have an “ori”, to amplify and mass-produce thevector in E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), and such, anda marker gene for selecting the transformed E. coli (e.g., adrug-resistance gene selected by a drug (e.g., ampicillin, tetracycline,kanamycin, or chloramphenicol)). For example, M13-series vectors,pUC-series vectors, pBR322, pBluescript, pCR-Script, and such can beused. Besides the vectors, pGEM-T, pDIRECT, pT7, and soon can also beused for the subcloning and excision of the cDNA as well. When a vectoris used to produce a protein of the present invention, an expressionvector is especially useful. When the expression vector is expressed,for example, in E. coli, it should have the above characteristics inorder to be amplified in E. coli. Additionally, when E. coli, such asJM109, DH5α, HB101, or XL1-Blue, are used as the host cell, the vectorshould have a promoter, e.g. lacZ promoter (Ward E S et al. (1989)Nature 341:544-546; Ward E S et al. (1992) FASEB J. 6:2422-2427), araBpromoter (Better M et al. (1988) Science 240:1041-1043), or T7 promoter,that can efficiently promote the expression of the desired gene in E.coli. Other examples of the vectors are pGEX-5X-1 (Pharmacia),“QIAexpress system” (QIAGEN), pEGFP, and pET (for this vector, BL21, astrain expressing T7 RNA polymerase, is preferably used as the host).

Further, the vector may comprise a signal sequence to secrete thepolypeptide. For producing the protein into the periplasm of E. coli,the pelB signal sequence (Lei S. P. et al. J. Bacteriol. 169:4379(1987)) may be used as the signal sequence for protein secretion. Forexample, the calcium chloride method or electroporation may be used tointroduce the vector into host cells.

As vectors used to produce the proteins of the present invention, forexample, expression vectors derived from mammals (e.g. pCDNA3(Invitrogen), pEF-BOS (Mizushima, S. and Nagata, S. Nucleic Acids Res.(1990) 18:5322), pEF, pCDM8), insect cells (e.g. “Bac-to-BAC baculovirusexpression system” (GIBCO-BRL), pBacPAK8), plants (e.g. pMH1, pMH2),animal viruses (e.g. pHSV, pMV, pAdexLcw), retroviruses (e.g. pZIPneo),yeasts (e.g. “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01), andBacillus subtilis (e.g. pPL608, pKTH50) can be mentioned other thanthose for E. coli.

In order to express proteins in animal cells, such as CHO, COS, andNIH3T3 cells, the vector must have a promoter necessary for expressionin such cells (e.g. SV40 promoter (Mulligan R C et al. (1979) Nature277:108-114), MMLV-LTR promoter, EF1α promoter (Mizushima, S. andNagata, S. (1990) Nucleic Acids Res. 18:5322), CMV promoter, etc.). Itis more preferable if the vector additionally had a marker gene forselecting transformants (for example, a drug resistance gene selected bya drug (e.g., neomycin, G418, etc.)). Examples of vectors with suchcharacteristics include PMAM, pDR2, PBK-RSV, pBK-CMV, pOPRSV, pOP13, andso on.

Furthermore, in order to stably express the gene and to amplify the copynumber in cells, the method using CHO cells deficient in nucleic acidsynthetic pathways as the host, incorporating into the CHO cells avector (such as pCHOI) having a DHFR gene that compensates for thedeficiency, and amplifying the vector with methotrexate (MTX) can beused. Furthermore, for transiently expressing a gene, the method thattransforms COS cells that have the gene for SV40 T antigen on thechromosome with a vector (such as pcD) having the SV40 replicationorigin can be mentioned. The replication origin may be that of apolyomavirus, adenovirus, bovine papilloma virus (BPV), and the like.Further, to amplify the gene copy number in the host cells, selectionmarkers such as the aminoglycoside transferase (APH) gene, thymidinekinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase(Ecogpt) gene, and the dihydrofolate reductase (dhfr) gene may becomprised in the expression vector.

DNA of the present invention can be expressed in animals by, forexample, inserting DNA of the invention into an appropriate vector andintroducing the vector into a living body by the retrovirus method,liposome method, cationic liposome method, adenovirus method, and so on.Thus, it is possible to perform gene therapy of diseases caused by amutation of the “ALEX1” gene of the present invention. The vectors usedin these methods include, but are not limited to, adenovirus vectors(e.g. pAdexlcw), retrovirus vectors (e.g. pZIPneo) and so on. Generaltechniques for gene manipulation, such as insertion of the DNA of theinvention into a vector, can be performed according to conventionalmethods (Sambrook, J. et al. (1989) Molecular Cloning 2nd ed.,5.61-5.63, Cold Spring Harbor Lab. press). Administration to the livingbody may be performed according the ex vivo method or the in vivomethod.

The present invention also provides a host cell into which a vector ofthe present invention has been introduced. The host cell into which thevector of the invention is introduced is not particularly limited. Forexample, E. coli, various animal cells, and such, can be used. The hostcell of the present invention can be used, for example, as a productionsystem to produce and express a protein of the present invention.Protein production systems include in vitro and in vivo systems. Suchproduction systems using eukaryotic cells or prokaryotic cells can begiven as in vitro production systems.

As eukaryotic host cells, for example, animal cells, plant cells, andfungi cells can be used. Mammalian cells, for example, CHO, COS, 3T3,myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells (e.g.platanna oocytes (Valle et al. (1981) Nature 291:358-340), and insectcells (e.g. Sf9, Sf21, Tn5) are known as animal cells. Among CHO cells,those deficient in the DHFR gene, dhfr-CHO (Urlaub, G. and Chasin, L. A.Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220) and CHO K-1 (Kao, F. T.and Puck, T. T. Proc. Natl. Acad. Sci. USA (1968) 60:1275-1281), areparticularly preferable. Among animal cells, CHO cells are particularlypreferable for mass expression. A vector can be introduced into a hostcell by, for example, the calcium phosphate method, the DEAE-dextranmethod, methods using cationic liposome DOTAP (Boehringer-Mannheim),electroporation, lipofection, etc.

As plant cells, for example, plant cells originating from Nicotianatabacum are known as protein producing systems and may be used as calluscultures. As fungal cells, yeast cells such as Saccharomyces, includingSaccharomyces cerevisiae, or filamentous fungi such as Aspergillus,including Aspergillus niger, are known.

Useful prokaryotic cells include bacterial cells. Bacterial cells suchas E. coli, for example, JM109, DH5α, HB101, and such, as well asBacillus subtilis are known.

These cells are transformed by desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. For example, culture medium such asDMEM, MEM, RPMI1640, or IMDM may be used with or without serumsupplements such as fetal calf serum (FCS) as culture medium for animalcells. The pH of the culture medium is preferably between about 6 and 8.Such cells are typically cultured at about 30 to 40° C. for about 15 to200 hr, and the culture medium may be replaced, aerated, or stirred ifnecessary.

Animal and plant hosts may be used for in vivo production. For example,desired DNA can be introduced into an animal or plant host. Encodedproteins are produced in vivo, and then recovered. These animal andplant hosts are included in the “host” of the present invention.

Animals to be used for the production system described above includemammals and insects. Mammals such as goats, pigs, sheep, mice, andcattle may be used (Vicki Glaser, SPECTRUM Biotechnology Applications(1993)). Alternatively, the mammals may be transgenic animals.

For instance, desired DNA may be prepared as a fusion gene with a genesuch as goat β casein gene that encodes a protein specifically producedinto milk. DNA fragments comprising the fusion gene are injected intogoat embryos, which are then introduced back to female goats. Proteinsare recovered from milk produced by the transgenic goats (i.e., thoseborn from the goats that had received the modified embryos) or fromtheir offspring. To increase the amount of milk containing the proteinsproduced by transgenic goats, appropriate hormones may be administered(Ebert K. M. et al. (1994) Bio/Technology 12:699-702).

Alternatively, insects, such as the silkworm, may be used. Baculovirusesinto which DNA encoding a desired protein has been inserted can be usedto infect silkworms, and the desired protein is recovered from the bodyfluid (Susumu M. et al. (1985) Nature 315:592-594).

As plants, for example, tobacco can be used. When using tobacco, DNAencoding a desired protein may be inserted into a plant expressionvector, such as pMON 530, which is introduced into bacteria, such asAgrobacterium tumefaciens. Then, the bacteria is used to infect tobacco,such as Nicotiana tabacum, and the desired polypeptide is recovered fromthe leaves (Ma, J K et al. (1994) Eur. J. Immunol. 24:131-138).

A protein of the present invention obtained as above may be isolatedfrom inside or outside of hosts (medium, etc.), and purified as asubstantially pure homogeneous protein. The method for protein isolationand purification is not limited to any specific method; in fact, anystandard method may be used. For instance, column chromatography,filters, ultrafiltration, salting out, solvent precipitation, solventextraction, distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, and recrystallizationmay be appropriately selected and combined to isolate and purify theprotein.

For chromatography, for example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, adsorption chromatography,and such may be used (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed. Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press (1996)). These chromatographiesmay be performed by liquid chromatographies such as HPLC and FPLC. Thus,the present invention provides highly purified proteins produced by theabove methods.

A protein may be optionally modified or partially deleted by treating itwith an appropriate protein-modifying enzyme before or afterpurification. For example, trypsin, chymotrypsin, lysylendopeptidase,protein kinase, glucosidase, and such are used as protein-modifyingenzymes.

The present invention also provides antibodies binding to a protein ofthe present invention. The antibodies of the present invention may takeany form, including monoclonal antibodies, as well as polyclonalantibodies. Furthermore, antiserum obtained by immunizing animals suchas rabbits and the like with a protein of the invention, all classes ofpolyclonal and monoclonal antibodies, as well as human and humanizedantibodies produced by genetic recombination are included.

A protein of the invention used as an antigen to obtain antibodies maybe derived from any animal species, but preferably it is from a mammalsuch as human, mouse, or rat, and more preferably from a human. Ahuman-derived protein may be obtained by using a nucleotide or aminoacid sequence disclosed herein.

A full-length protein or a partial peptide thereof may be used as anantigen in the present invention. A partial peptide may be, for example,an amino (N)-terminus or carboxy (C)-terminus fragment of the protein.Alternatively, a peptide having a transmembrane region of “ALEX1” andsuch may be used. Herein, an “antibody” is defined as an antibody thatreacts with either the full length or a fragment of the protein.

A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector used to transform a host cell asdescribed herein. The desired protein or its fragment may be recoveredfrom the outside or inside of host cells by any standard method, and maybe used as the antigen. Alternatively, cells expressing the protein ortheir lysates, or a chemically synthesized protein may be used as anantigen. Preferably, short peptides are used as antigens byappropriately binding to carrier proteins such as keyhole limpethemocyanin, bovine serum albumin, or ovalbumin.

Any mammal may be immunized with the antigen, but preferably, thecompatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha, or Primates areused.

Animals of Rodentia include, for example, mice, rats, and hamsters.Animals of Lagomorpha include, for example, rabbits. Animals of Primatesinclude, for example, monkeys of Catarrhini (old world monkeys) such asMacaca fascicularis, rhesus monkeys, sacred baboons, or chimpanzees.

Methods for immunizing animals with antigens are well known.Intraperitoneal injection or subcutaneous injection of antigens is usedas a standard method. More specifically, antigens may be diluted andsuspended in an appropriate amount with phosphate buffered saline (PBS),physiological saline, etc. If desired, the antigen suspension may bemixed with an appropriate amount of a standard adjuvant, such asFreund's complete adjuvant, made into an emulsion, and then administeredto mammals. Preferably, this is followed by several administrations ofthe antigen mixed with an appropriate amount of Freund's incompleteadjuvant every 4 to 21 days. An appropriate carrier may also be used forimmunization. After the above immunization, the serum is examined for anincrease of the amount of desired antibodies by a standard method.

Polyclonal antibodies raised against a protein of the present inventionmay be prepared by collecting blood from the immunized mammal afterconfirming the increase of desired antibodies in the serum, and byseparating serum from the blood by any conventional method. Serumcontaining a polyclonal antibody may also be used as a polyclonalantibody, or if necessary, the fraction containing the polyclonalantibody may be isolated from the serum. For example, fractions thatrecognize only a protein of the present invention are obtained by usingaffinity columns to which the present protein is coupled, and by furtherpurifying the fraction using a protein A or G column, immunoglobulin Gor M may be prepared.

To prepare monoclonal antibodies, immune cells are collected from amammal immunized with an antigen after checked for an increase of thelevel of the desired antibodies in the serum as described above, andthese cells are subjected to cell fusion. The immune cells used for cellfusion are preferably obtained from the spleen. The other parent cellfused with the above immune cell is preferably a mammalian myeloma cell,and more preferably, a myeloma cell that has acquired a special featurethat can be used for selecting fusion cells by a drug.

The above immune cell and myeloma cell may be fused by basically anystandard method, such as those described in literature (Galfre G. andMilstein C. Methods Enzymol. 73:3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as the HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for a period of timethat is sufficient to allow all cells except the desired hybridoma(non-fused cells) to die, usually from several days to several weeks.Then, standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

Besides the above method in which a nonhuman animal is immunized with anantigen for preparing a hybridoma, human lymphocytes such as thoseinfected by the EB virus may be immunized with a protein,protein-expressing cells, or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells that are capableof indefinite division, such as U266, to yield a hybridoma producing adesired human antibody capable of binding to a protein of the invention(Unexamined Published Japanese Patent Application (JP-A) No. Sho63-17688).

Subsequently, the hybridomas thus obtained are transplanted into theperitoneal cavity of a mouse from which the ascites is collected. Themonoclonal antibodies thus obtained can be purified by, for example,ammonium sulfate precipitation or column chromatography using a proteinA or protein G column, a DEAE ion exchange column, an affinity columnand such to which a protein of the invention is coupled. An antibody ofthe invention can be used not only for purifying and detecting a proteinof the invention, but also as a candidate for an agonist or antagonistof a protein of the present invention. An antibody of this invention maybe a human antibody or humanized antibody.

For example, transgenic animals having a repertory of human antibodygenes may be immunized with a protein, protein expressing cells, ortheir lysates as antigen. Antibody producing cells are collected fromthe animals, and fused with myeloma cells to obtain hybridoma, fromwhich human antibodies against the protein can be prepared (seeWO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, andWO96-34096).

Alternatively, an immune cell that produces antibodies, such as animmunized lymphocyte, may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Such monoclonal antibodies can also be recombinantly prepared usinggenetic engineering techniques (see, for example, Borrebaeck C. A. K.and Larrick J. W. Therapeutic Monoclonal Antibodies, published in theUnited Kingdom by MacMillan Publishers LTD (1990)). A recombinantantibody can be prepared by cloning DNA encoding the antibody from animmune cell such as a hybridoma or an immunized lymphocyte producing theantibody, inserting this into an appropriate vector, and introducing thevector into a host cell. The present invention also encompassesrecombinant antibodies prepared as described above.

An antibody of the present invention may be a fragment of an antibody ormodified antibody, so long as it binds to one or more of the proteins ofthe invention. For instance, the antibody fragment may be Fab, F(ab′)₂,Fv, or single chain Fv (scFv), in which Fv fragments from H and L chainsare linked by an appropriate linker (Huston J. S. et al. (1988) Proc.Natl. Acad. Sci. U.S.A. 85:5879-5883). More specifically, an antibodyfragment may be generated by treating an antibody with an enzyme such aspapain or pepsin. Alternatively, a gene encoding the antibody fragmentmay be constructed, inserted into an expression vector, and expressed inan appropriate host cell (see, for example, Co M. S. et al. (1994) J.Immunol. 152:2968-2976; Better M. and Horwitz A. H. (1989) MethodsEnzymol. 178:476-496; Pluckthun A. and Skerra A. (1989) Methods Enzymol.178:497-515; Lamoyi E. Methods Enzymol. (1986) 121:652-663; Rousseaux J.et al. (1986) Methods Enzymol. 121:663-669; Bird R. E. and Walker B. W.(1991) Trends Biotechnol. 9:132-137).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides suchmodified antibodies. A modified antibody can be obtained by chemicallymodifying an antibody. These modification methods are conventional inthe field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody comprising a variable region derived from a nonhumanantibody and the constant region derived from a human antibody, or as ahumanized antibody comprising the complementarity determining region(CDR) derived from a nonhuman antibody, the framework region (FR)derived from a human antibody, and the constant region, by usingwell-known methods.

Obtained antibodies may be purified to homogeneity. Any standard methodprotein separation and purification method may be used for antibodyseparation and purification. For example, chromatographies such asaffinity chromatography, filters, ultrafiltration, salting out,dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing,and such may be appropriately combined to isolate and purify theantibody (Antibodies: A Laboratory Manual. Ed Harlow and David Lane,Cold Spring Harbor Laboratory, 1988) However, the methods are notlimited thereto. The concentration of the obtained antibody may bedetermined by measuring absorbance, by enzyme-linked immunosorbent assay(ELISA), etc.

Columns used for affinity chromatography include, protein A column andprotein G column. For example, Hyper D, POROS, Sepharose F. F.(Pharmacia), and such may be mentioned as columns using protein Acolumns.

Chromatographies other than affinity chromatography are, for example,ion exchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, adsorption chromatography,and so on (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). These chromatographies can be conductedusing liquid chromatographies such as HPLC, and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), orimmunofluorescence may be used to measure the antigen binding activityof an antibody of the invention. In ELISA, an antibody of the presentinvention is immobilized on a plate, a protein of the invention isapplied, and then a sample containing a desired antibody, such asculture supernatant of antibody producing cells or a purified antibody,is applied. Then, a secondary antibody labeled with an enzyme such asalkaline phosphatase that recognizes the primary antibody is applied,and the plate is incubated. After washing, an enzyme substrate, such asp-nitrophenyl phosphate, is added to the plate, and the absorbance ismeasured to evaluate the antigen binding activity of the sample. Afragment of a protein, such as the C-terminus fragment, may be used asthe protein. BIAcore (Pharmacia) may be used to evaluate the activity ofan antibody according to the present invention.

The above methods allow the detection or measurement of a protein of theinvention, by exposing an antibody of the invention to a sample assumedto contain the protein of the invention, and detecting or measuring theimmune complex formed by the antibody and the protein. Because themethod of detection or measurement of a protein according to theinvention can specifically detect or measure a protein, the method maybe useful in a variety of experiments in which the protein is used. Themethod is useful in testing for Alzheimer's disease or cancer asdescribed below.

The present invention also provides a polynucleotide comprising at least15 nucleotides that is complementary to DNA encoding the human “ALEX1”protein (SEQ ID NO: 1 or 2) or to the complementary strand thereof.Polynucleotides of this invention are useful in, for example, to detector amplify the proteins of the invention, to detect the expression ofDNA, or regulate the expression. Herein, the detection of DNA includesthe detection of mutations in the DNA.

“Complementary strand” herein refers to one strand of a double strandnucleic acid comprising A:T (U for RNA) and G:C base pairs, when viewedagainst the other strand. Furthermore, “complementary” means not onlywhen a nucleotide sequence is completely complementary to a continuousnucleotide sequence with at least 15 nucleotides, but also when there isa homology of at least 70% or more, preferably 80% or more, morepreferably 90% or more, and much more preferably 95% or more at thenucleotide sequence level. Homology can be determined by using thealgorithm described herein.

Such a polynucleotide includes a polynucleotide having at least 15nucleotides that hybridizes to DNA encoding the human “ALEX1” protein(SEQ ID NO: 1 or 2), or to its complementary strand. Preferably, thepolynucleotide specifically hybridizes to DNA encoding the human “ALEX1”protein (SEQ ID NO: 1 or 2), or to its complementary strand.“Specifically hybridize” means that the polynucleotide does notsignificantly hybridize to DNA encoding another protein under ordinaryhybridization conditions, preferably under the above-described stringentconditions.

Such polynucleotides include probes and primers used for the detectionand amplification of DNA encoding a protein of the present invention,probes and primers used for the detection of the expression of the DNA,nucleotides and nucleotide derivatives (for example, antisenseoligonucleotides, ribozymes, or DNA encoding them, etc.) used forregulating the expression of a protein of the present invention.Furthermore, such polynucleotides can be used in the preparation of DNAchips or microarray. Polynucleotides of this invention include DNA andRNA, and also sense nucleotides and antisense nucleotides.

If the polynucleotide is used as a primer, the 3′ region thereof may bethe complementary site, and restriction enzyme recognition sites, tagsequences, and such may be attached to the 5′ region.

Antisense oligonucleotides comprise, for example, an antisenseoligonucleotide that hybridizes with any portion of the nucleotidesequence of SEQ ID NO: 1 or 2. The antisense oligonucleotide ispreferably an antisense of a continuous sequence comprising at least 15nucleotides or more within the nucleotide sequence of SEQ ID NO: 1 or 2.More preferably, the above continuous sequence comprising at least 15nucleotides or more contains a translation initiation codon.

A derivative or modified form of an antisense oligonucleotide may alsobe used. The latter form may be prepared by modifying an antisenseoligonucleotide with lower alkylphosphonates such as methylphosphonateor ethylphosphonate, or with phosphorothioate, or phosphoroamidate.

The antisense oligonucleotide is not restricted to one in which allnucleotides are complementary to the corresponding nucleotides within agiven region of DNA or mRNA. As long as it can specifically hybridizewith the nucleotide sequence of SEQ ID NO: 1 or 2, it may have one ormore nucleotide mismatches.

A derivative of an antisense oligonucleotide of the present inventionmay act on cells producing a protein of the invention and bind to DNA ormRNA encoding the protein, and then, it may inhibit the expression ofthe protein of the invention by inhibiting its transcription ortranslation, or by promoting the degradation of mRNA, and therebyinhibiting the function of the protein.

A derivative of an antisense oligonucleotide of the present inventionmay be mixed with an appropriate base that is inactive against thederivative, and used as a medicine for external application, such as anointment or poultice.

If necessary, it may be mixed with an excipient, isotonizing agent,solubilizing agent, stabilizer, preservative, pain-killer, or the like,and prepared as a tablet, powder, granule, capsule, liposome capsule,injectable solution, liquid formulation, nose drops, freeze-dried agent,etc. The above may be achieved according to standard methods.

For treating patients, a derivative of an antisense oligonucleotide ofthe present invention may be, for example, directly applied to theaffected area of a patient, or administered into blood vessels so as tofinally reach the affected area. Moreover, the derivative may beencapsulated in antisense-encapsulating materials such as liposomes,poly-L-lysine, lipid, cholesterol, lipofectin, or their derivatives inorder to increase durability and/or membrane permeability.

Dose of the derivative of the antisense oligonucleotide of the presentinvention may be appropriately adjusted depending on the patient'sconditions, and a favorable amount such as 0.1 to 100 mg/kg, or more,preferably 0.1 to 50 mg/kg, may be administered.

As an antisense oligonucleotide of the present invention inhibits theexpression of a protein of the invention, it is useful as an inhibitorof a biological activity of the protein of the invention. An inhibitorof expression comprising an antisense oligonucleotide of the presentinvention is useful due to its ability to inhibit a biological activityof a protein of the invention.

The antibodies of this invention and the polynucleotides of thisinvention are useful for testing cancer and Alzheimer's disease. Theexpression of “ALEX1” gene of this invention was significantly low incancer cells. This indicates that “ALEX1” can be used for testingcancer. For example, it is possible to test the presence of cancer cellsand progress of cancer by detecting the protein of this invention ormRNA encoding this protein in a test sample. Also, since “ALEX1” proteininteracts with IDE that is involved in the β-amyloid metabolism, and inaddition interacts with PS-1, it is thought to be deeply involved withthe onset and progress of Alzheimer's disease. Therefore, “ALEX1” may beused for testing and diagnosing Alzheimer's disease.

In this invention, “testing Alzheimer's disease or cancer” is not onlytesting patients expressing symptoms of Alzheimer's disease or cancercaused by a mutation of the ALEX1 gene, but also testing ALEX1 geneexpression level and genetic mutation performed to decide whether thesubject is likely to have Alzheimer's disease or cancer due to anaberration in ALEX1 gene expression level or genetic mutation. That is,even when symptoms have not yet been expressed, the danger ofcontracting Alzheimer's disease or cancer is thought to be very highwhen there is an aberration in ALEX1 gene expression, or when a mutationoccurs in one of the ALEX1 alleles.

The test for Alzheimer's disease or cancer of this invention can beperformed using, for example, antibodies that bind to the protein ofthis invention, or polynucleotides comprising at least 15 nucleotidesthat are complementary to DNA encoding the protein of this invention, orto its complementary strand. When the antibodies of this invention andthe polynucleotides of this invention are used as test reagents, theycan be combined appropriately with stabilizers, preservatives, and salt,solutes such as buffers, water, and solvents such as physiologicalsaline.

One of the test methods for Alzheimer's disease and cancer of thisinvention is a method comprising detecting the expression level of DNAencoding a protein of this invention in a test sample. Such a testmethod includes methods comprising (a) contacting the above-mentionedpolynucleotide with a patient-derived RNA sample, and (b) detectingbinding of the polynucleotide to the RNA sample. Such tests can beperformed, for example, by Northern hybridization or RT-PCR. A test ofthis invention that uses RT-PCR specifically comprises (a) synthesizingcDNA from a patient-derived RNA sample, (b) performing a polymerasechain reaction using the synthesized cDNA as a template and theabove-mentioned polynucleotide as a primer, and (c) detecting the DNAamplified by polymerase chain reaction. Northern hybridization andRT-PCR can be performed by well-known genetic engineering techniques.Also, detection by DNA chips or DNA microarrays are also possible.

The test method for Alzheimer's disease or cancer of this invention maycomprise detecting the level of a protein of this invention present in apatient-derived test sample. Such tests can be performed, for example,using antibodies against the protein of this invention. Specifically, atest using an antibody of this invention comprises (a) contacting anantibody of this invention with a patient-derived protein sample, and(b) detecting binding of the antibody to the protein sample. Proteinscan be detected by immunoprecipitation using the antibodies of thisinvention, Western blotting, immunohistochemistry, ELISA, and such.

Specifically, these tests may specify cancer and Alzheimer's diseasefoci by investigating expression by methods such as immunohistologicalstaining or in situ hybridization on tissues collected by biopsy.Decrease in expression of ALEX1 suggests the possibility of onset and/orprogress of cancer or Alzheimer's disease. Diseases such as cancer andAlzheimer's are thought to occur due to various reasons. For example,when a decrease in ALEX1 expression is confirmed in cancer, since theactivation of the JNK pathway is expected, these tests may be used fordiagnosis in performing tailor-made therapy where treatment targetingthis pathway is performed.

In addition, the test for Alzheimer's disease or cancer of thisinvention may be performed by detecting a mutation in the protein ofthis invention, or a mutation in the DNA encoding this protein. Sincethe ALEX1 gene is thought to be involved with the onset and/or progressof Alzheimer's disease and cancer, mutations of the protein or the DNAsuggest the danger of onset and progress of Alzheimer's disease orcancer.

A mutation of a protein of this invention includes structural andfunctional mutations. For example, a structural mutation of a protein ina patient-derived protein sample can be tested by using an antibody ofthis invention and comparing the molecular weight of the protein with aprotein derived from a healthy person using Western blotting. Also, itis possible to detect mutations of a protein of this invention by usingas indexes, changes in protein modification, changes in binding ofproteins or antibodies that bind to the protein of this invention. Forthese tests, it is possible to use, for example, ELISA,immunoprecipitation, pull-down method, and such that use an antibodyagainst a protein of this invention.

The test for Alzheimer's disease or cancer of this invention may also beperformed by detecting the binding of a protein of this invention withthe IDE protein, PS-1 protein, or JIP-1 protein. Otherwise, it may beperformed by detecting binding of a protein of this invention with p0071(plakophilin-4), SART-1, MSP58, ATRX, CSA2 (RED protein), p68 (RNAhelicase/ATPase), OS-9, ZNF189, KIAA1221, α-Actinin4, or ZIP kinase.Inhibition of the binding between a protein of this invention and such aprotein is thought to contribute to the onset and progress ofAlzheimer's disease or cancer. Binding of proteins can be evaluated, forexample, by ELISA, immunoprecipitation, and pull-down method, using anantibody against a protein of this invention.

For detecting a mutation in DNA encoding a protein of this invention, anucleotide sequence of cDNA encoding the protein of this invention, andoligonucleotide (probe and primer) complementary to the nucleotidesequence of a genomic DNA sequence (including endogenous transcriptionregulatory sequence) or to its complementary strand may be used.Incidentally, testing a mutation includes a testing that specifiespatients (carriers) having a mutation in one of the ALEX1 alleles.

When used as a primer, polynucleotides are normally 15 to 100 bp, andpreferably 17 to 30 bp. There are no limitations on the primer as longas it can amplify at least a portion of the ALEX1 gene region or aregion that regulates its expression. Examples of such regions include,for example, exons, introns, promoters, and enhancer regions of theALEX1 gene.

On the other hand, as a probe, the polynucleotide normally comprises atleast 15 bp or longer if it is a synthetic polynucleotide.Double-stranded DNA obtained from a clone that has been inserted into avector such as plasmid DNA may also be used as a probe. There are nolimitations on the probe as long as it is complementary to thenucleotide sequence of at least a portion of the ALEX1 gene or theregion regulating its expression, or to its complementary strand. Theregion to which the probe hybridizes includes, for example, the exon,intron, promoter, and enhancer regions of the ALEX1 gene. When used asprobes, the polynucleotide or the double stranded DNA are labeledappropriately, and then used. The labeling methods are, for example,phosphorylating the 5′-end of polynucleotide with ³²P using T4polynucleotide kinase, or incorporating a substrate nucleotide labeledwith biotin, fluorescent dye, isotope such as ³²P, and such, and using arandom hexamer oligonucleotide as a primer and DNA polymerase such asKlenow enzyme (random priming method).

One embodiment of a method for detecting mutations of the ALEX1 gene isto directly determine the nucleotide sequence of the ALEX1 gene of apatient. For example, using the above-mentioned nucleotide as a primer,and DNA isolated from a patient suspected to have a disease caused bymutation of ALEX1 as a template, a portion or whole of the patient'sALEX1 gene (for example, regions including exon, intron, promoter, andenhancer) is amplified by, for example, PCR (Polymerase Chain Reaction),and its nucleotide sequence is determined. By comparing this to thesequence of a normal person's ALEX1 gene, diseases caused by a mutationof the ALEX1 gene can be tested.

As the testing method of this invention, various methods are usedbesides the method of directly determining the nucleotide sequence ofDNA derived from patients. In one embodiment, the method comprises, (a)preparing a DNA sample from a patient, (b) amplifying thepatient-derived DNA using the polynucleotide of this invention as aprimer, (c) dissociating the amplified DNA into single-stranded DNA, (d)separating the dissociated single stranded DNA on a non-denaturing gel,and (e) comparing the mobility of the separated single-stranded DNA onthe gel with a control from a healthy person.

An example of such methods is PCR-SSCP (single-strand conformationpolymorphism) method (Cloning and polymerase chainreaction-single-strand conformation polymorphism analysis of anonymousAlu repeats on chromosome 11. Genomics. Jan. 1, 1992; 12(1): 139-146,Detection of p53 gene mutations in human brain tumors by single-strandconformation polymorphism analysis of polymerase chain reactionproducts. Oncogene. Aug. 1, 1991; 6(8): 1313-1318, Multiplefluorescence-based PCR-SSCP analysis with postlabeling, PCR MethodsAppl. Apr. 1, 1995; 4(5): 275-282). This method is relatively facile andhas the advantage of requiring a small amount of samples. Therefore, itis especially preferable when screening many DNA samples. Its principlesare as follows. When a double-stranded DNA fragment is dissociated intosingle strands, each strand forms an independent higher-order structureaccording to its nucleotide sequence. When this dissociated DNA strandis electrophoresed in a polyacrylamide gel that does not contain adenaturant, depending on the difference in each higher-order structure,complementary single-stranded DNA having the same chain length move to adifferent position. Higher order structure of such single-stranded DNAchanges even with replacement of a single nucleotide, and indicatesdifferent mobility in polyacrylamide gel electrophoresis. Therefore, bydetecting this change in mobility, existence of a mutation in the DNAfragment such as point mutation, deletion, or insertion can be detected.

Specifically, first the whole ALEX1 gene, or a portion of it, isamplified by PCR and such. Normally, the amplified range preferably hasapproximately 200 to 400 bp. Also, the amplified range includes allexons and all introns of the ALEX1 gene and also, promoters andenhancers of the ALEX1 gene. During gene fragment amplification by PCR,synthesized DNA fragments are labeled by performing PCR using a primerlabeled with isotopes such as ³²P or with fluorescent dyes, biotin, andsuch, or by adding a substrate nucleotide labeled with isotopes such as³²P, or with fluorescent dyes, biotin, and such into the PCR solution.Otherwise, labeling can be performed by adding a substrate nucleotidelabeled with isotopes such as ³²P, or with fluorescent dyes, biotin, andsuch to a synthesized DNA fragment using Klenow enzyme, and such, afterPCR. The labeled DNA fragment obtained this way is denatured by heatingand such, and electrophoresis is performed using a polyacrylamide gelthat does not contain denaturants such as urea. Here the conditions forDNA fragment separation can be improved by adding an appropriate amount(approximately 5 to 10%) of glycerol to the polyacrylamide gel. Also,electrophoretic conditions change with properties of each DNA fragment,but normally, it is performed at room temperature (20 to 25° C.). When afavorable separation cannot be obtained, the temperature that gives themost appropriate mobility is tested between 4 to 30° C. Afterelectrophoresis, mobility of the DNA fragment is detected byautoradiography using an X-ray film, a scanner detecting fluorescence,and such, and then analyzed. When a band having a difference in mobilityis detected, this band is directly cut out from the gel, thenre-amplified by PCR, and by direct sequencing, the existence of amutation can be confirmed. Also, even when labeled DNA is not used, bystaining the gel after electrophoresis with ethidium bromide or silverstaining and such, bands can be detected.

Another embodiment of the testing method of this invention includes (a)preparing a DNA sample from a patient, (b) amplifying thepatient-derived DNA using a polynucleotide of this invention as aprimer, (c) cleaving the amplified DNA, (d) separating the DNA fragmentsaccording to their size, (e) hybridizing the detectably labeled probeDNA of this invention to the separated DNA fragment, and (f) comparingthe size of the detected DNA fragment with a control of a healthyperson.

Examples of such methods are methods using restriction fragment lengthpolymorphism (RFLP), PCR-RFLP, and such. Normally, restriction enzymesare used as enzymes to cleave DNA. Specifically, when a mutation existsat the restriction enzyme recognition site, or when there is anucleotide insertion or deletion in the DNA fragment formed byrestriction enzyme treatment, the size of the fragment formed afterrestriction enzyme treatment changes compared to that of a healthyperson. This portion containing the mutation is amplified by PCR, and bytreatment with each restriction enzyme, these mutations can be detectedas differences in band mobility after electrophoresis. Otherwise,chromosomal DNA is treated with these restriction enzymes and afterelectrophoresis, by performing Southern blotting using the probe DNA ofthis invention, the presence or absence of mutation can be detected. Therestriction enzyme to be used can be selected appropriately depending oneach mutation. In this method, besides genomic DNA, RNA prepared from apatient is made into cDNA by reverse transcriptase and after cleaving itin its original form by a restriction enzyme, Southern blotting can beperformed. Also, using this cDNA as a template, a portion or whole ofthe ALEX1 gene can be amplified by PCR, and after cleaving them withrestriction enzyme, their difference in mobility can be investigated.

Furthermore, instead of using DNA prepared from a patient, a similardetection is possible using RNA as well. Such a method comprises (a)preparing a RNA sample from a patient, (b) separating the prepared RNAaccording to size, (c) hybridizing the polynucleotide of this inventionthat has a detectable label as a probe to the separated RNA, and (d)comparing the size of the detected RNA with a control from a healthyperson. An example of a specific method comprises electrophoresing RNAprepared from a patient, performing Northern blotting using apolynucleotide of this invention as a probe, and detecting differencesof mobility.

Another embodiment of the test methods of this invention comprises (a)preparing a DNA sample from a patient, (b) amplifying thepatient-derived DNA using the polynucleotide of this invention as aprimer, (c) separating the amplified DNA on a gel in which theconcentration of DNA denaturant gradually rises, and (d) comparingmobility of the separated DNA on the gel compared to a control of ahealthy person.

An example of such a method is denaturant gradient gel electrophoresis(DGGE). The whole ALEX1 gene, or a portion of it, is amplified by PCRusing the primer of this invention, and such. This is thenelectrophoresed in a polyacrylamide gel in which the concentration of adenaturant such as urea gradually becomes higher as the material moves,and this is compared to that of a healthy person. For a DNA fragment inwhich a mutation exists, the DNA fragment becomes single stranded at aposition of lower denaturant concentration, and the rate of movementbecomes extremely slow. Therefore, by detecting this difference inmobility, the presence or absence of a mutation can be detected.

Besides these methods, for the purpose of detecting mutations only at aparticular position, allele specific oligonucleotide (ASO) hybridizationcan be used. An oligonucleotide containing a sequence in which amutation is thought to exist is prepared, and when this is hybridizedwith sample DNA, if a mutation exists, the efficiency of hybridformation decreases. This can be detected by Southern blotting or by amethod that utilizes the property of quenching by intercalation of aspecialized fluorescent reagent into a gap in the hybrid, or such amethod. Also, detection by ribonuclease A mismatch cleavage method ispossible. Specifically, the whole ALEX1 gene, or a portion of it, isamplified by PCR and such, and the product is hybridized with labeledRNA prepared from ALEX1 cDNA and such inserted in a plasmid vector, andsuch. In the portion in which a mutation exists, the hybrid becomes asingle-stranded structure, therefore, this portion is cleaved byribonuclease A, and by detecting this with autoradiography, and such,existence of a mutation can be detected.

A protein of the invention may be useful for screening for a compoundthat binds to the protein. Specifically, the protein may be used in amethod of screening for the compound binding to the protein of theinvention, such a method comprising the steps of exposing the protein ofthe present invention to a test sample expected to contain a compoundbinding to the protein, and selecting a compound having the activity ofbinding to the protein.

Proteins of the invention used for screening may be recombinant ornatural proteins, or partial peptides. For example, screening can beperformed using a partial peptide lacking the transmembrane domain, or apartial peptide containing a transmembrane domain, and such. Compoundsthat bind to the transmembrane domain of ALEX1 protein may regulateintracellular localization of the protein by interfering withtransmembrane domain function. Proteins used for screening may be in theform expressed on the surface of a cell, or in the form of a membranefraction. Samples tested include, but are not limited to, cell extracts,cell culture supernatants, products of fermentation microorganisms,marine organism extracts, plant extracts, purified or crude preparationsof proteins, peptides, non-peptide compounds, synthetic low-molecularweight compounds, and natural compounds. A protein of the presentinvention contacted with a test sample may be brought into contact withthe test sample, for example, as a purified protein, as a solubleprotein, in the form attached to a carrier, a fusion protein with otherproteins, in the form expressed on the cell membrane, or as a membranefraction.

Various methods known to one skilled in the art may be used as thescreening method of, for example, a protein that binds to a protein ofthe present invention using a protein of the present invention. Such ascreening can be carried out, for example, by the immunoprecipitationmethod. Specifically, the method can be carried out as follows. A geneencoding a protein of this invention is expressed by inserting the geneinto vectors for foreign gene expression such as pSV2neo, pcDNA I, andpCD8, and expressing the gene in animal cells, etc. Any generally usedpromoter may be employed for the expression, including the SV40 earlypromoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3.Academic Press, London, p. 83-141 (1982)), EF-1 α promoter (Kim, D M etal. Gene 91, p. 217-223 (1990)), CAG promoter (Niwa, H. et al. Gene 108,p. 193-200 (1991)), RSV LTR promoter (Cullen, B R, Methods in Enzymology152, p. 684-704 (1987)), SR α promoter (Takebe Y et al., Mol. Cell.Biol. 8, p. 466-472 (1988)), CMV immediate early promoter (Seed B andAruffo A, Proc. Natl. Acad. Sci. USA 84, p. 3365-3369 (1987)), SV40 latepromoter (Gheysen D and Fiers W, J. Mol. Appl. Genet. 1, p. 385-394(1982)), Adenovirus late promoter (Kaufman R J et al., Mol. Cell. Biol.9, p. 946-958 (1989)), HSV TK promoter, etc. Transfer of a foreign geneinto animal cells for expression therein can be performed by any of thefollowing methods, including the electroporation method (Chu, G. et al.,Nucl. Acid Res. 15, 1311-1326 (1987)), the calcium phosphate method(Chen, C. and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)), theDEAE dextran method (Lopata, M. A. et al. Nucl. Acids Res. 12, 5707-5717(1984); Sussman, D. J. and Milman, G. Mol. Cell. Biol. 4, 1642-1643(1985)), the lipofectin method (Derijard, B. Cell. 7, 1025-1037 (1994);Lamb, B. T. et al. Nature Genetics 5, 22-30 (1993)), Rabindran, S. K. etal. Science 259, 230-234 (1993)), etc. A protein of this invention canbe expressed as a fusion protein having a recognition site for amonoclonal antibody whose specificity has been established byintroducing the recognition site (epitope) into the N- or C-terminal ofa protein of this invention. For this purpose, a commercialepitope-antibody system can be utilized (Jikken Igaku (ExperimentalMedicine) 13, 85-90 (1995)). Vectors that are capable of expressingfusion proteins with β-galactosidase, maltose-binding protein,glutathione S-transferase, green fluorescence protein (GFP) and such,via a multi-cloning site are commercially available.

To minimize the alteration in properties of a protein of this inventiondue to fusion protein formation, a method for preparing a fusion proteinby introducing only a small epitope portion comprising several to tenamino acid residues has been reported. For example, the epitopes ofpolyhistidine (His-tag), influenza agglutinin (HA), human c-myc, FLAG,Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein(T7-tag), human herpes simplex virus glycoprotein (HSV-tag), E-tag(epitope on the monoclonal phage), and such, and monoclonal antibodiesto recognize these epitopes can be utilized as the epitope-antibodysystem for screening for proteins binding to the protein of thisinvention (Jikken Igaku (Experimental Medicine) 13, 85-90 (1995)).

In immunoprecipitation, immune complexes are formed by adding theseantibodies to the cell lysate prepared using suitable detergents. Thisimmune complex comprises a protein of this invention, a protein capableof binding to the protein, and an antibody. The immunoprecipitation canalso be performed using an antibody to a protein of this invention,besides antibodies to the above-described epitopes. An antibody to aprotein of this invention can be prepared by, for example, inserting agene encoding a protein of this invention into an appropriate expressionvector of E. coli to express it in the bacterium, purifying the proteinthus expressed, and immunizing rabbits, mice, rats, goats, chicken, andsuch, with the purified protein. The antibody can also be prepared byimmunizing the above-described animals with synthetic partial peptidesof a protein of this invention.

Immune complexes can be precipitated using, for example, Protein ASepharose and Protein G Sepharose when the antibody is a murine IgGantibody. In addition, when the protein of this invention is prepared asa fusion protein with the epitope of, for example, GST, and such, theimmune complex can be precipitated using a substance that specificallybinds to these epitopes, such as glutathione-Sepharose 4B, and such,giving the same result as in the case where the antibody for the proteinof this invention is used.

Immune precipitation, in general, may be carried out according to, orfollowing the method described in literature (Harlow, E. and Lane, D.:Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, NewYork (1988)).

SDS-PAGE is generally used for the analysis of immunoprecipitatedproteins. Bound proteins can be analyzed based on the molecular weightsof proteins using a gel of an appropriate concentration. In this case,although proteins bound to the protein of this invention, are in generalhardly detectable by the usual protein staining method, such asCoomassie staining and silver staining, the detection sensitivity can beimproved by culturing cells in a medium containing radio isotope-labeled³⁵S-methionine and ³⁵S-cysteine to incorporate radiolabelsbiosynthetically, and detecting the labeled proteins. Once the molecularweight of the protein is determined, the desired protein can be purifieddirectly from SDS-polyacrylamide gel and sequenced.

Isolation of a protein that binds to a protein of the present inventionusing the protein may be carried out by, for example, using theWest-Western blotting method (Skolnik E. Y. et al. (1991) Cell65:83-90). Specifically, a cDNA library is constructed from cells,tissues, or organs (for example, tissues from ovary, heart, testis,prostate, brain, spleen, skeletal muscle, colon, and so on) in which aprotein binding to a protein of the present invention is expected to beexpressed, by using phage vectors (λgt11, ZAP, etc.). Then, this isexpressed on LB-agarose, transferred to a filter membrane, which isreacted with a purified labeled protein of the invention. The plaquesexpressing proteins that bind to the protein of the invention can beidentified by detecting the label. The protein of the invention may belabeled by a method utilizing the binding between biotin and avidin, ora method utilizing an antibody that specifically binds to the protein ofthe present invention, or a peptide or polypeptide (e.g. GST and such)that is fused to the protein of the present invention. Methods usingradioisotope or fluorescence and such may also be used.

Alternatively, in another embodiment of the method for screening of thepresent invention, a two-hybrid system utilizing cells may be used(Fields S. and Sternglanz R. (1994) Trends Genet. 10:286-292; Dalton S.and Treisman R. (1992) Characterization of SAP-1, a protein recruited byserum response factor to the c-fos serum response element, Cell68:597-612; “MATCHMAKER Two-Hybrid System”, “Mammalian MATCHMAKERTwo-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (products ofClontech); “HybriZAP Two-Hybrid Vector System” (Stratagene)). Thetwo-hybrid system can be used as follows: (1) a protein of the presentinvention or a partial peptide thereof is fused to the SRF DNA bindingregion or GAL4 DNA binding region and expressed in yeast cells; (2) acDNA library, which expresses proteins as fusion proteins with VP16 orGAL4 transcription activating regions, is prepared from cells expectedto express proteins binding to the protein of the present invention; (3)the library is introduced to above mentioned yeast cells; and (4)library-derived cDNA are isolated from the positive clones detected(positive clones can be confirmed by activation of reporter genes due tothe binding of the present protein and the binding protein expressed inthe yeast cell). The protein encoded by the cDNA can be obtained byintroducing the isolated cDNA into E. coli and expressing it. Thus, aprotein binding to a present protein or genes thereof can be prepared.For example, in addition the HIS3 gene, Ade2 gene, LacZ gene, CAT gene,luciferase gene, PAI-1 (Plasminogen activator inhibitor type1) gene, andso on, can be mentioned as reporter genes used in the 2-hybrid system,but are not restricted thereto. In addition to yeast, mammalian cellsand such can be used for the 2-hybrid screening.

Alternatively, a protein binding to a protein of the present inventioncan be screened by affinity chromatography. For example, a protein ofthe invention is immobilized on a carrier of an affinity column, and atest sample, in which a protein capable of binding to the protein of theinvention is presumed to be expressed, is applied to the column. Thetest sample used herein may be a cell extract, cell lysate, etc. Afterloading the test sample, the column is washed, and a protein bound tothe protein of the invention can be obtained.

The DNA encoding the protein may be obtained by analyzing the amino acidsequence of the obtained protein, synthesizing oligo DNA based on thesequence information, and screening a cDNA library using the DNA as theprobe.

A biosensor utilizing the surface plasmon resonance phenomenon may beused as a means for detecting or measuring bound proteins. When such abiosensor is used, the interaction between a protein of the inventionand a test protein can be observed at real-time as a surface plasmonresonance signal, using only a minute amount of proteins withoutlabeling (for example, BIAcore, Pharmacia) Therefore, it is possible toevaluate the binding between a protein of the invention and a testcompound using a biosensor such as BIAcore.

In addition, methods for isolating not only proteins, but also compoundsbinding to the proteins of the invention (including agonists andantagonists) are known in the art. Such methods include, for example,the method of screening for a binding molecule by contacting synthesizedcompounds or natural substance libraries, or random phage peptidedisplay libraries with an immobilized protein of the invention, and thehigh-throughput screening method using the combinatorial chemistrytechnique (Wrighton, N. C. et al., Small peptides as potent mimetics ofthe protein hormone erythropoietin, Science, 1996, 273 p 458-64; VerdineG. L., The combinatorial chemistry of nature, Nature, 1996, 384, p11-13; Hogan J. C. Jr., Directed combinatorial chemistry, Nature, 1996,384, p 17-9).

This invention also provides a screening method for compounds thatregulate binding between the protein of this invention and the IDEprotein. This screening method comprises, (a) contacting the protein ofthis invention with the IDE protein in the presence of a test sample,(b) detecting the binding between these proteins, and (c) selecting acompound having activity to promote or inhibit the binding between theseproteins, compared to when the detection is made in the absence of thetest sample.

This invention also provides a screening method for compounds thatregulate the binding between the protein of this invention and the PS-1protein. This screening method comprises, (a) contacting the protein ofthis invention with the PS-1 protein in the presence of a test sample,(b) detecting the binding between these proteins, and (c) selecting acompound having activity to promote or inhibit the binding between theseproteins, compared to when the detection is made in the absence of thetest sample.

This invention also provides a screening method for compounds thatregulate binding between the protein of this invention and the JIP-1protein. This screening method comprises, (a) contacting the protein ofthis invention with the JIP-1 protein in the presence of a test sample,(b) detecting the binding between these proteins, and (c) selecting acompound having activity to promote or inhibit the binding between theseproteins, compared to when the detection is made in the absence of thetest sample.

This invention also provides a screening method for compounds thatregulate the binding between the protein of this invention and a proteinselected from the group consisting of p0071 (plakophilin-4), SART-1,MSP58, ATRX, CSA2 (RED protein), p68 (RNA helicase/ATPase), OS-9,ZNF189, KIAA1221, α-Actinin4, and ZIP kinase. This screening methodcomprises, (a) contacting the protein of this invention with a proteinselected from the group consisting of p0071 (plakophilin-4) SART-1,MSP58, ATRX, CSA2 (RED protein), p68 (RNA helicase/ATPase) OS-9, ZNF189,KIAA1221, α-Actinin4, and ZIP kinase in the presence of a test sample,(b) detecting the binding between these proteins, and (c) selecting acompound having activity to promote or inhibit the binding between theseproteins, compared to when the detection is made in the absence of thetest sample.

Each protein used for these screenings are not restricted by its form aslong as it has the ability to form a complex or has a binding ability.It does not have to be a full length protein, and it can be a mutant ora partial peptide. It may also be a natural protein or a recombinantprotein. It may be a fusion protein with another peptide. These proteinsmay be prepared similarly to the above-mentioned case relating toscreening of compounds that bind to the protein of this invention. Also,human derived proteins, or proteins derived from other animals can beused as proteins, such as IDE protein, PS-1 protein, and JIP-1 protein,that are contacted with the protein of this invention. Samples testedinclude, but are not limited to, cell culture supernatants, products offermentation microorganisms, marine organism extracts, plant extracts,prokaryotic cell extracts, eukaryotic single cell extracts, animal cellextracts, libraries thereof, purified or crude preparations of proteins,peptides, non-peptide compounds, synthetic low-molecular weightcompounds, and natural compounds. A protein used for the screening maybe used, for example, as a purified protein, as a soluble protein, inthe form attached to a carrier, a fusion protein with other proteins, inthe form expressed on the cell membrane, or as a membrane fraction.

This type of screening can be performed, for example, by a two-hybridmethod using yeast or animal cells. Specifically, DNA encoding theprotein of this invention, and DNA encoding a protein contacted with theprotein of this invention, such as IDE, PS-1, or JIP-1, are fused toeither a sequence encoding a transcription activation domain or a DNAbinding sequence of an appropriate transcription factor such as GAL4,intracellularly co-expressed with a GAL4 luciferase reporter vector andsuch, and a promoter or inhibitor substance can be screened using thisluciferase activity as an index.

It is also possible to perform screening using, for example,immunoprecipitation. The protein of this invention and a protein such asIDE, PS-1, or JIP-1, contacted with the protein of this invention areincubated in the presence of a test sample, a complex is collected withan antibody against one of the proteins or with an antibody against atag fused to the protein, and such, and then by detecting the otherprotein using antibodies and such against that protein, binding with theprotein of this invention can be evaluated. If the other protein islabeled with a different tag, and such, detection can be easily done.One of the proteins is bound to a support, then the other protein isbound, and then, a test sample is applied. By detecting whether thebound protein dissociates, the effect of the test sample can beinvestigated. Also, it is possible to perform screening using ELISA.

Screening can also be performed by utilizing surface plasmon resonanceas indicated in the screening of proteins that bind to the protein ofthis invention, high-throughput screening utilizing combinatorialchemistry, etc.

This invention also provides a screening method that uses as an index,inhibition of MKK7/JNK-mediated signal transduction by expression of aprotein of this invention. This screening method comprises (a)contacting a test sample with a cell expressing the protein of thisinvention, (b) detecting MKK7/JNK-mediated signal transduction in thecell, and (c) selecting a compound having activity to promote or inhibitthe signal transduction. There are no particular limitations on the testsample.

The proteins of this invention, MKK7, JNK, and such may be endogenous orthose externally introduced for expression, but to confirm specificityof compounds obtained by screening to ALEX1, it is preferable toexternally introduce the ALEX1 gene using a cell that does not haveendogenous ALEX1 expression. Most epithelial cancer cells match thiscriterion. By comparison with cells to which the ALEX1 gene had not beenintroduced, specificity towards ALEX1 can be confirmed. For example,HeLaS3 cells used in Example 10 are thought to be preferable forscreening as they have no endogenous expression of ALEX1 and have anendogenous expression of MKK7. Also, to avoid too much complexity ofscreening system, a cell in which components of the JNK pathway and JIP1are expressed endogenously, and the JNK pathway operates responding tostimuli, is preferred. For this type of example, there are no particularlimitations, but MCF7 breast cancer cells (Monno, S. et al.,Endocrinology 2000, 141(2):544-50) or KB3 cells (Stone, A. A. &Chambers, T. C., Exp. Cell Res. 2000, 254(1):110-9) may be used.Especially in the former, since activation of the JNK pathway by IGF-1stimulus has been reported, introduction of MEKK1 is not necessary, anda screening system may be constructed with relative ease.

It is also considered preferable to use, for example, human epithelialcancer cell line derived from oral cavity, KB (ATCC #CCL-17). KB cellsinclude MKK7 and JNK, and the JNK pathway is activated by TNFα(Moriguchi, T. et al. (1997) EMBO J. 16, 7045-7053). The JNK pathway ofKB cells can be also activated with IL-1 (Krause, A. et al. (1998) J.Biol. Chem. 273, 23681-23689). A549 cells can also be used. Expressionof ALEX1 cannot be detected by the Northern blotting, nor by RT-PCR inA549 cells (see Examples). The JNK pathway of this cell can be activatedby EGF (Bost, F. et al. (1997) J. Biol. Chem. 272, 33422-33429). Anexample of other cells is ovarian cancer cell line SK-OV-3, in which theJNK pathway can be activated by Cisplatin treatment, but is not limitedto this example (Persons et al. (1999) Clin. Cancer Res. 5, 1007-1014).

Substances affecting MKK7/JNK-mediated signal transduction can bescreened, for example, as shown in Example 10, by detectingc-Jun-dependent transcription. Specifically, a vector expressing thec-Jun protein that has been fused with an appropriate DNA bindingprotein such as GAL4, and a reporter vector in which a reporter genesuch as luciferase is linked downstream of a binding sequence of the DNAbinding protein are introduced into cells expressing the protein of thisinvention, and by detecting the reporter in the presence or absence of atest sample, screening can be performed using the effect of the testsample on promotion or inhibition of c-Jun-dependent transcription bythe protein of this invention as an index. As another method,ATF2-dependent reporter activity reported to be activated by JNK(Adamson, A. L. et al., J. Virol. 2000, 74(3):1224-33) can be detected.It is also possible to detect phosphorylation of c-Jun or ATF2 proteinthrough MKK7 using antibodies. Specifically, for c-Jun, detection ispossible by sandwich ELISA using an antibody against phosphorylated(N-terminal) c-Jun and an antibody against another portion of c-Jun (oranti-tag antibody). A similar system can be used in the case of ATF2.

Compounds that bind to the proteins of this invention, compounds thatregulate binding between the proteins of this invention and proteinsthat bind to them, and compounds that regulate MKK7/JNK-mediated signaltransduction, isolable by the above-mentioned screening, become, forexample, candidates of drugs that promote or inhibit the activity of theproteins of this invention. Therefore, such a compound can be andapplied to the treatment of diseases caused by an expressional orfunctional abnormality, and such, of a protein of this invention anddiseases that are treatable by regulating the activity of a protein ofthis invention.

It may also be possible to utilize genes encoding the proteins of thisinvention or their expression regulatory regions to screen for compoundsthat may adjust the expression (including transcription and translation)of these genes in vivo or in cells. This screening may be used, forexample, for screening for candidate compounds for therapeutic andprophylactic agents for cancer and Alzheimer's disease.

This screening may be performed by the method comprising, (a) contactinga test sample with a cell that endogenously expresses the DNA of thisinvention, (b) detecting the expression, and (c) selecting a compoundhaving activity to promote or inhibit the expression compared to whenthe cell is not contacted with the test sample (control). Transcriptionand translation are included in this DNA expression.

For example, screening of the desired compound can be performed bycultivating cells expressing the ALEX1 gene with a test sample,detecting expression (including transcription and translation) of thegene by mRNA detection methods such as Northern analysis and RT-PCR, orprotein detection methods such as Western blotting, immunoprecipitation,and ELISA, or by methods in which these are improved, and selectingcompounds that promote or inhibit expression of the gene compared towhen the test sample is not added.

There are no particular limitations on the cells used for screening, butfrom the viewpoint of cancer therapy where induction of ALEX1 expressionis expected to lead to treatment, screening of compounds that promoteexpression of ALEX1 using various cancer cell lines in which theexpression of ALEX1 is decreased, such as cervical adenocarcinoma cellline HeLa, lung carcinoma cell line A549, non-small cell lung carcinomacell line ABC-1, and cervical adenocarcinoma cell line C-33A can beconsidered. On the contrary when decrease in expression is theobjective, screening can be performed using glioblastoma cell line A172,osteosarcoma cell line TE-85, or normal fibroblastic cell line MRC5, andsuch.

In methods to detect mRNA such as Northern analysis and RT-PCR, forexample, a polynucleotide containing at least 15 nucleotidescomplementary to DNA comprising the nucleotide sequence of SEQ ID NO: 1or 2 or to its complementary strand may be used as a probe or a primer.In methods to detect proteins such as Western blotting,immunoprecipitation, and ELISA, an antibody of this invention can beused.

Screening of compounds that regulate the expression of the gene of thisinvention in vivo or within a cell can also be done by a method thatuses the activation or inactivation of expression regulatory region ofthe gene of this invention as an index. This screening may be performedby the method comprising (a) contacting a test sample with a cell intowhich a vector having a reporter gene operably linked downstream of anendogenous transcription regulatory sequence of the DNA of thisinvention has been introduced, (b) detecting expression of the reportergene within the cell, and (c) selecting a compound having activity topromote or inhibit expression of the reporter gene compared to when thecell is not contacted with the test sample (control).

Here, “endogenous transcription regulatory sequence” refers to sequencesregulating transcription of the DNA in cells naturally maintainingexpression of the DNA of this invention. Such sequences includepromoters, enhancers, and/or repressors. For these sequences, forexample, DNA in the upstream region of a gene encoding the protein ofthis invention may be used. For example, a DNA fragment spanning severalkb upstream from the transcription initiation site (or translationinitiation codon) of a gene encoding the protein of this invention, isconsidered to contain the endogenous transcription regulatory sequenceof the gene mentioned above. By joining this fragment with a reportergene, it is possible to place expression of the reporter gene undertranscriptional control of a gene encoding the protein of thisinvention. It is possible to measure transcription regulating activityby introducing deletions or mutations appropriately to the upstreamregion, and to identify the sequence involved in transcriptionalregulation and to use that fragment. Currently, many transcriptionregulatory sequences that bind to transcription factors involved invarious transcriptional regulations are known. Such known transcriptionregulatory sequences could be found in the upstream region of a geneencoding the protein of this invention, and regarded to be involved inthe endogenous transcription regulatory sequence. Normally, multiplesequences regulating gene transcription exist on one gene, and in thescreening of this invention, any one sequence or a combination ofsequences may be used. Endogenous transcription regulatory sequences maybe made as chimeras with other promoters. Chimeric promoters are oftenused for testing transcriptional regulation. Other promoters used toproduce chimeric promoters include, for example, a minimum promoterderived from SV40 early promoter. “Operably linked” indicates that thetranscription regulatory sequence and a reporter gene are linked so thatthe reporter gene linked downstream may be expressed in response toactivation of the transcription regulatory sequence.

Specifically, for example, by screening a genomic DNA library using thenucleotide sequence of SEQ ID NO: 1 or 2 or a portion of it as a probe,transcription regulatory region (promoter, enhancer, etc.) of the DNA ofthis invention is cloned, then an expression vector in which this isinserted upstream of an appropriate reporter gene (chloramphenicolacetyl transferase gene, luciferase gene, etc.) is prepared, and then,this is introduced to a mammalian cell. Next, a test sample is contactedwith the cell line, reporter activity is detected, and by selectingcompounds that increase or decrease reporter activity compared toreporter activity in cells that are not contacted with the test sample,compounds that may regulate the expression of the gene of this inventionwithin a cell can be screened. Because this screening detects expressionof the DNA of this invention using reporter activity as an index, it isvery convenient, compared to direct detection such as theabove-mentioned Northern analysis.

The compounds that regulate the expression of the genes of thisinvention, isolated by these screenings become candidates of drugstoward various diseases caused by an aberrant expression of the gene ofthis invention. Especially, use as a drug for preventing and treatingdiseases such as Alzheimer's disease and cancer is anticipated.

ALEX1 is thought to suppress the onset and progress of cancer. InAlzheimer's disease, it is thought to suppress the onset or progress byguiding IDE, which is thought to contribute to the breakdown of amyloidpeptides, to the sites of amyloid production. Compounds that promote thebinding between ALEX1 and JIP-1, compounds that inhibit MKK7/JNK signaltransduction, and compounds that elevate expression of ALEX1 maycontribute to the prevention and treatment of cancer. Similarly, inAlzheimer's disease, compounds that promote the binding between ALEX1and IDE, compounds that promote the binding between ALEX1 and PS-1, andcompounds that elevate the expression of ALEX1 are thought to contributeto the prevention and treatment of Alzheimer's disease. Furthermore,since JNK is involved in apoptosis, compounds obtained from thescreenings of this invention may be applied to apoptosis-relateddiseases. JNK pathway is activated by various stresses, however, itsinvolvement in apoptosis differs according to the cell type. In oneexample, in apoptosis of myocardial cells injured by high oxygentension, the JNK pathway is suggested to function protectively byinhibiting TNF production (Minamino, T. et al., Proc. Natl. Acad. Sci.USA 1999, 96(26):15127-32). On the other hand, mouse fibroblast cellslacking c-Jun are resistant to apoptosis caused by drugs that alkylateDNA, and this has been explained to be due to the involvement of c-Junin inducing the expression of CD95L (FASL) caused by an alkylating agent(Kolbus, A. et al., Mol. Cell Biol. 2000 January; 20(2): 575-82).Therefore, in a combination of a stimulus and a cell system in whichactivation of JNK promotes apoptosis, cell protection due to aninhibition of JNK pathway can be expected. On the contrary, in acombination that suppresses apoptosis, induction of cell death due toinhibition of JNK pathway can be expected.

In addition, since compounds that inhibit the binding between ALEX1 andJIP-1, compounds that promote MKK7/JNK signal transduction, andcompounds that decrease the expression of ALEX1 can inhibit themechanism that is normally regulated negatively by ALEX1, they may beused for organ regeneration, and such. Also, compounds that inhibit thebinding between ALEX1 and IDE, compounds that inhibit the bindingbetween ALEX1 and PS-1, and compounds that decrease the expression ofALEX1 are useful for pathological analysis of Alzheimer's disease andfor producing animal models of Alzheimer's disease.

Also, since proteins such as p0071 (plakophilin-4), SART-1, MSP58, ATRX,CSA2 (RED protein), p68 (RNA helicase/ATPase), OS-9, ZNF189, KIAA1221,α-Actinin4, and ZIP kinase that interact with ALEX1 are suggested to berelated to Alzheimer's disease and/or cancer, compounds that regulatethe binding between ALEX1 and these proteins are also expected to have autility as drugs for preventing or treating diseases such as Alzheimer'sdisease or cancer.

Compounds that may be isolated by the screenings of this inventionbecome candidates of drugs regulating the activity or expression of theproteins of this invention. Thus, therapeutic application is possiblefor diseases caused by expressional aberrations, functional aberrations,and such of the protein of this invention, and diseases treatable byregulating the activity and expression of a protein of this invention.Substances in which a part of the structure of compounds that may beisolated by the screening method of this invention has been modified byan addition, deletion, and/or substitution are included in the compoundsthat bind to the proteins of this invention.

When using a compound that can be isolated by the screenings of thisinvention as a pharmaceutical agent for humans and other mammals, suchas mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle,monkeys, baboons, and chimpanzees, and also for chicken, the protein orthe isolated compound can be directly administered or can be formulatedusing known pharmaceutical preparation methods. For example, accordingto the need, the drugs can be taken orally as sugarcoated tablets,capsules, elixirs and microcapsules or non-orally in the form ofinjections of sterile solutions or suspensions with water or any otherpharmaceutically acceptable liquid. For example, the compounds can bemixed with pharmacologically acceptable carriers or medium,specifically, sterilized water, physiological saline, plant-oil,emulsifiers, solvents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives and binders, in a unit dose formrequired for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier, such as oil, can also beincluded in the above ingredients. Sterile composites for injections canbe formulated following normal drug implementations using vehicles suchas distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80™ andHCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; a pain-killer such as procaine hydrochloride;a stabilizer such as benzyl alcohol, phenol; and an anti-oxidant. Theprepared injection is filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administer apharmaceutical agent to patients, for example as intraarterial,intravenous, subcutaneous injections and also as intranasal,transbronchial, intramuscular, percutaneous, or oral administrations.The dosage varies according to the body-weight and age of a patient, andthe administration method, but one skilled in the art can suitablyselect the dosage. If said compound can be encoded by DNA, the DNA canbe inserted into a vector for gene therapy to perform the therapy. Thedosage and method of administration vary according to the body-weight,age, and symptoms of a patient, but one skilled in the art can selectthem suitably.

Although varying according to the subject, target organ, symptoms, andmethod of administration, the dose of a compound that binds to a proteinof this invention, a compound that inhibits or promotes the activity ofa protein of this invention, a compound that inhibits or promotes theexpression of the protein, and such, which can be isolated by thescreening of this invention, are generally in the range of about 0.1 to100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to20 mg per day for adults (body weight: 60 kg) in the case of an oraladministration.

Although varying according to the subject, target organ, symptoms, andmethod of administration, a single dose of a compound for parenteraladministration is preferably, for example, when administeredintravenously to normal adults (60 kg body weight) in the form ofinjection, in the range of about 0.01 to 30 mg, preferably about 0.1 to20 mg, and more preferably about 0.1 to 10 mg per day. Doses convertedto 60 kg body weight or per body surface area can be administered toother animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in vitro interaction of ALEX1 with IDE.

In vitro translated ALEX1 protein was incubated with eitherIDE-Flag-immobilized beads or control beads. After washing the beads,the bound proteins were separated by SDS-PAGE and observed byautoradiography. In vitro translated luciferase used as a negativecontrol ([³⁵S]Lucifer) did not bind to IDE-Flag beads. The [³⁵]ALEX1 and[³⁵S]luciferase used for the reactions (containing 10% of proteins addedto the reactions) are shown for comparison (lanes “input”). In thisfigure, lane M shows [¹⁴C]methylated protein molecular weight markers.

FIG. 2 shows multiple alignment of the amino acid sequences of the humanALEX1, ALEX2 (GenBank Accession No. BAA25438), and ALEX3.

The amino acid sequence for ALEX3 is the translation of the putativehuman cDNA sequence (257925) in the TIGR database. Amino acid sequenceswere aligned by ClustalW, and homologous amino acids were shaded usingBoxshade 3.21 program. Black residues are identical amino acids. Armmotifs are boxed.

FIG. 3 shows multiple alignment of the amino acid sequences of the humanALEX1, ALEX2, and ALEX3 (continued from FIG. 2).

FIG. 4 shows Northern blot analysis of ALEX1 and ALEX2 mRNA expression.

Northern blot containing 2 μg poly (A)⁺ RNA from various human adulttissues (Clontech) was hybridized with the probe against the codingregion of the ALEX1 and ALEX2. Molecular size markers are shown on theleft.

FIG. 5 shows tissue-specific expression pattern of ALEX1 and ALEX2 mRNAin normal tissues compared with tumor samples.

cDNA prepared from various tumor samples and corresponding normaltissues (Multiple Tissue cDNA Panels, Clontech) were used as templatesfor PCR with the primers specific for ALEX1 and ALEX2. PCR products werethen analyzed by electrophoresis using 2% agarose/EtBr gel. N, normaltissues. Lung carcinomas: C₁, Human Lung Carcinoma (LX-1); C₂, HumanLung Carcinoma (GI-117). Prostate carcinoma: C, Human ProstaticAdenocarcinoma (PC3). Colon carcinomas: C₁, Human Colon Adenocarcinoma(CX-1); C₂, Human Colon Adenocarcinoma (GI-112). Pancreatic carcinoma:C, Human Pancreatic Adenocarcinoma (GI-103). Ovarian carcinoma: C, HumanOvarian Carcinoma (GI-102). DNA size markers (φX174 digested withHaeIII) are shown in lane M.

FIG. 6 shows Northern blot analysis of ALEX1 in human tumor and normalovaries.

Northern blot contains 20 μg of total RNA prepared from each of tumorand corresponding normal tissues excised at the same operational site(Human Ovarian Tumor Blot, Invitrogen). Donors: 1—serouscystadenocarcinoma of left ovary (age—48 years old); 2—serouscystadenocarcinoma (30 years); 3—granulosa-theca cell tumor (42 years);4—adenocarcinoma (28 years). T, tumor tissue; N, normal tissue.

FIG. 7 shows expression of ALEX1 and ALEX2 mRNA in human normal comparedwith transformed cell lines.

cDNA prepared from various human cell lines and normal tissues were usedas templates for PCR with the same primers combinations used in theabove figure. PCR products were then analyzed by electrophoresis using2% agarose/EtBr gel. Normal tissues (Human MTC Panel, Clontech): brain,heart, kidney, and spleen. Cell lines of human origin: MRC-5, normalfetal lung diploid fibroblast cell line; KMS-6, normal diploid fetalfibroblast cell line; KMST-6T, neopastically transformed cell linederived from KMS-6; TE-85, malignant osteosarcoma cell line; t-HUE2,immortal cell line established from endothelial cell line ECV304; U-2OS, osteosarcoma cell line; HeLa, cervix adenocarcinoma cell line; A549,lung carcinoma cell line; ABC-1, non-small cell lung carcinoma cellline; A172, malignant glioma cell line; C-33 A, cervical carcinoma cellline; CMV-Mj-HEL-1, CMV-transformed embryo lung fibroblast cell line.DNA size markers (φX174 digested with HaeIII) are shown in lane M.

FIG. 8 shows expression analysis of ALEX1 mRNA during mouse embryodevelopment.

Northern blot containing 2 μg poly (A)⁺ RNA from whole mouse embryo atdifferent stages of development (Clontech) was hybridized to the probeprepared based on the ALEX1-homologous sequence found in mouse ESTdatabase. Molecular size markers are shown on the left.

FIG. 9 shows the analysis of interaction of JIP-1 fragments with ALEX1protein.

JIP-1 deletion constructs were transformed into PJ69-4A yeast carryingpODB-80-ALEX1 using a plasmid to which a Gal-4 transcription activationdomain was ligated. This was then plated on plates that lacked Leu, Trp,and His, and contained 1 mM 3-AT. Growth on -Leu-Trp-His plate indicatesinteraction. In parallel, the yeast were plated onto -Leu-Trp plate toconfirm successful transformation. Fragment of amino acid numbers 258 to711 indicates the smallest JIP-1 clone (19A3) found originally in theyeast two-hybrid screen.

FIG. 10 shows inhibition of c-Jun-dependent transcription activation byALEX1.

The c-Jun-dependent reporter gene pG5-Luc (encoding firefly luciferaseprotein) (500 ng) together with the Renilla luciferase-encoding plasmidpRL-TK (50 ng) as the internal control, and expression vectors for Gal4(25 ng), Gal4-cJun (25 ng), MEKK1 (50 ng), and ALEX1 were transfectedinto HeLa S3 cells as indicated. Luciferase activity was measured 42 hpost-transfection. The values shown on the vertical axis represent therelative level of firefly luciferase to the Renilla luciferase activityused as the internal control. The assay was performed in duplicates.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below with referenceto Examples, but it is not to be construed as being limited thereto. Allreferences cited herein are incorporated into this description. Primersused in Examples are as follows: ALEX1 specific primers:

ALEX1 specific primers: F1, GTGCTCGGGTTAAGAGATTTGTC; (SEQ ID NO: 4) F2,TCACAATGATCTGGTGGTG; (SEQ ID NO: 5) F3, CAACATGACTGTGACTAATC; (SEQ IDNO: 6) R1, AGCTCCTTTCACAGTCTC; (SEQ ID NO: 7) R2,ACCCAACCATTACAACCAACATCAG; (SEQ ID NO: 8) R3,GGCCATGTTGTAGCTGGAGCCCTGGTGC; (SEQ ID NO: 9) ALEX2 specific primers: F4,TAGCAGCACCTACCAAGGTAG; (SEQ ID NO: 10) F5, TGCCTTGCTTCAGAAATCTG; (SEQ IDNO: 11) R4, CCCAGTTCGTCTACTTCAACT; and (SEQ ID NO: 12) R5,CTTCCACACTGCAAAATCATG. (SEQ ID NO: 13)

Sequences were determined on both strands of double stranded-DNA usingthe ABI dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, andan ABI Prism 377 Genetic Analyzer (Perkin-Elmer/Applied Biosystems). Thenucleotide sequences obtained were assembled and analyzed using AutoAssembler DNA Sequence Assembly Software (Applied Biosystems) Homologieswith known nucleic acids and proteins registered in the GenBank and EMBLdatabases were analyzed using the BLAST algorithm (Altschul S. F. et al.(1990) J. Mol. Biol. 215, 403-410). Amino acid sequences were comparedusing the BLASTP and PROSITE programs (Altshul, S. F. et al. (1997)Nucleic Acids Res. 25, 3389-3402; Bairoch, A. et al. (1997) NucleicAcids Res. 25, 189-196). Motif searches were performed with Pfam at theSanger Center, UK. Prediction of protein transmembrane domains wasperformed with SOSUI program (Hirokawa, T. et al. (1998) Bioinformatics14, 378-379). Amino acid sequence alignment was obtained with Clustal W(Thompson, J. D. et al. (1994) Nucleic Acids Res. 22, 4673-4680).

Example 1 cDNA Cloning and Protein Analysis

The yeast two-hybrid system was used to isolate proteins that interactwith IDE. Yeast two-hybrid screening was performed with the strainPJ69-4A (James, P. et al. (1996) Genetics 144, 1425-1436) carrying threereporter genes, HIS3, ADE, and LacZ under the control of GAL4 promoter.Mutation-introduced full-length insulin-degrading enzyme IDE (H108Q; themutant lacking catalytic activity but preserving substrate-bindingactivity) was linked to Gal4-DB (DNA-binding domain of Gal4) in frame,and cloned into pODB-80 vector. This was used as a bait plasmid. Yeastcells were transformed with the bait plasmid and the expression offusion proteins was confirmed by Western blot. Then, the yeast cellscarrying the bait plasmid were transformed with a prey plasmid, thepACT2-human brain cDNA library (Clontech). Double transformants carryingboth of the bait and prey were directly selected on synthetic medium(SD) lacking Leu, Trp, and His plus 1 mM 3-aminotriazole (3-AT). Growingclones were then selected on SD lacking Leu, Trp, His, and adenine, andassayed for β-galactosidase activity by filter assay. Plasmid DNA wasextracted from positive colonies using Plasmid Mini Kit from Qiagen andelectroporated into E. coli DH5α strain. Human brain-derived insertsobtained were sequenced with GAL4-AD specific primer.

During this process, several candidate cDNA were obtained from normalhuman brain library. Sequencing of the 1290 bp cDNA insert from clone B4revealed that the sequence has an 879-bp-long open reading frame (ORF),encoding a polypeptide of 293 amino acids. Analysis of the predictedamino acid sequence against Pfam database identified within it two 42amino acid armadillo/beta-catenin-like repeats. The insert from clone B4already contained polyA-sequence at its 3′-end. Therefore, 5′-RACE wasemployed to obtain the rest of the sequence using human heart cDNA as atemplate.

The 5′-RACE was performed using Advantage cDNA Polymerase Mix (Clontech)with primer R3 and pAP3neo human heart cDNA library (Takara) as atemplate. The touch-down PCR was conducted under the conditions asfollows: 94° C. for 1 min, for 1 cycle; 94° C. for 30 sec, 72° C. for 5min for 5 cycles; 94° C. for 30 sec, 70° C. for 5 min for 5 cycles; 94°C. for 20 sec, 68° C. for 5 min for 25 cycles; 72° C. for 7 min for 1cycle; and the temperature was held at 4° C. The PCR-product obtainedwas purified via electrophoresis, cloned into the vector pCR2.1-TOPO(Invitrogen), and sequenced using T7 primer, M13 primer, and a series ofinsert-specific primers.

The largest amplified product contained additional 851 bp to thesequence of B4 toward 5′ direction, bringing total length of thesequence to 2141 bp together with the B4 sequence (named ALEX1). Thenucleotide sequence and predicted amino acid sequence of this ALEX1 cDNAare shown in SEQ ID NOS: 1 and 3, respectively. Because of possibilityof errors introduced during RACE-PCR, B4 cDNA was cloned also byhybridization-based screening of cDNA library.

Namely, Human Testis cDNA library cloned in pCMV-SPORT vector (GibcoBRL) was divided into 36 pools each containing 3072 clones and griddedonto Nylon filters. The pools were screened by PCR with F1/R1 primersand ALEX1 cDNA-positive filters were hybridized to the 208-bp probegenerated by PCR using same primers. The probe was radiolabeled with[α-³²P] dCTP (3000 Ci/mmol) (Amersham) using the Megaprime DNA LabelingSystem (Amersham). Inserts from respective positive clones weresequenced.

As compared with the sequence obtained in the above RACE method (SEQ IDNO: 1), the sequence of clone 144D10 having the largest insert length(2124 bp) obtained through this screening (SEQ ID NO: 2) differs in the44 nucleotides at the 5′-end, and its 45^(th) (G) corresponds to 51^(st)(G) of SEQ ID NO: 1. There is a single nucleotide insertion of (A) atthe 77^(th) nucleotide, and three nucleotides at positions 129 to 131(AGT) are deleted. There are differences in the 3′-end as well:substitution of the 2130^(th) nucleotide of A to C; 2132^(nd) nucleotideof A to G; and there is no poly(A). Other parts including the codingregions are identical. As will be shown later on, the divergence in thelatter nucleotide sequence occurs exactly at the junction of exon 1 andexon 2 of the gene.

E. coli containing the ALEX1 cDNA clone isolated by screening the humantestis-derived cDNA library has been deposited as “pCMV-SPORT-ALEX1” inthe following depository.

(a) Name and address of depository

-   -   Name: National Institute of Bioscience and Human-Technology,        Advanced Industrial Science and Technology, Ministry of Economy,        Trade and Industry    -   (Old name: National Institute of Bioscience and        Human-Technology, Agency of Industrial Science and Technology)    -   Address: 1-1-3 Higashi, Tsukuba, Ibaraki, Japan (Zip code        305-8566)        (b) Date of deposition (Date of original deposition):    -   Feb. 25, 2000        (c) Accession number: FERM BP-7056

The putative initiation codon of the ALEX1 cDNA sequence (SEQ ID NO: 1)is the ATG starting from position 372 and located within a nucleotidesequence adequate for translation initiation signal (ACCATGG) (Kozak, M.(1984) Nucleic Acids Res. 12, 857-873). The first in-frame stop codon(TAA) was identified at nucleotide 1731, predicting a protein product of453 amino acids with a calculated molecular weight of 49,178 and acalculated isoelectric point (pI value) of 9.56. The polyadenylationsignal AATAAA is located 19 bp upstream of the polyadenylation startingsite (SEQ ID NO: 1).

Sequence analysis of the ALEX1 protein revealed in addition to theabove-described two Arm repeats, an ATP/GTP-binding site at 162^(nd) to169^(th) amino acids. Potential phosphorylation sites present in ALEX1protein include eight potential protein kinase C sites and fivepotential casein kinase II sites. The SOSUI algorithm predicts atransmembrane domain at the N-terminus of the protein (1^(st) to 25^(th)amino acids). The transmembrane domain contains four putativemyristoylation sites. At the C-terminus, ALEX1 contains microbodiestargeting signal.

Example 2 Interaction of ALEX1 with IDE

As described above, the yeast two-hybrid screen with full-length IDEmutant (H108Q) as a bait identified clone B4 corresponding to C-terminalpart of ALEX1 protein (amino acid numbers 161 to 453). This promptedadditional studies to determine whether IDE interacts with full-lengthALEX1 and also whether the interaction between two proteins is direct.For this purpose, the full-length ALEX1- and IDE-encoding cDNA weresubcloned into the E. coli expression vector pGEX-5X to express ALEX1and IDE as fusion proteins with GST. Both proteins were found in E. coliinsoluble fraction. ALEX1 was solubilized from the inclusion bodiesusing a denaturant. Obtained protein, however, was likely to bemisfolded as it interacted with several unrelated ³⁵S-labeled proteins(data not shown). Such difficulties associated with the expression ofmammalian proteins in E. coli were widely known. To overcome this, theinventors switched to the insect expression system. Flag-tagged IDEmutant (H108Q) was expressed in Sf9 cells and purified using agarosebeads onto which M2-anti-Flag antibody was immobilized.

Specifically, IDE mutant (H108Q) cDNA fused to the Flag epitope at the5′-end was cloned into the insect cell expression vector pIZ/V5-His(InsectSelect System, Invitrogen). The construct was transfected intothe insect cell line Sf9. Stably transfected cells were selected by thegrowth in a medium containing Zeocin at 0.4 mg/ml.

For in vitro binding assays, the gene-introduced cells were collected,washed in PBS, resuspended in lysis buffer (containing 50 mM Tris-HCl,pH 7.2, 150 mM NaCl, 1% Triton X-100 and Complete protease inhibitorcocktail, Boehringer Mannheim), sonicated to disrupt the cells, andcentrifuged 10 min at 15,000×g. Supernatant after the centrifugation waspurified through the affinity column in which the M2 anti-Flag antibodywas immobilized (Sigma). Specifically, the supernatant after thecentrifugation was applied onto the column, and then the column waswashed with the binding buffer (containing 20 mM Tris-HCl, pH 7.2, 150mM NaCl, 1 mM EDTA, 0.1% Tween-20, and Complete protease inhibitorcocktail). Bound IDE-Flag protein was eluted with Flag peptide (Sigma)at 0.1 mg/ml and dialyzed against the binding buffer to remove the Flagpeptide.

On the other hand, the biosynthesis of full-length ALEX1 protein wasperformed to produce the protein labeled with [³⁵]-methionine in rabbitreticulocyte lysates. For binding experiments, 5 μl of ³⁵S-labeled ALEX1or luciferase (negative control) was mixed with 2.5 μg of IDE in 0.4 mlof the binding buffer and incubated for 2 h at 4° C. Then 15 μl of theanti-Flag M2 antibody-immobilized affinity beads was added andincubation continued for 2.5 h at 4° C. The beads were washed five timesin the binding buffer. Bound protein was eluted with 0.1 mg/ml Flagpeptide, resolved by SDS-PAGE, stained with Coomassie blue and analyzedwith a Phosphoimager (BAS2000, Fuji).

The interaction of the both proteins was examined as above, ALEX1 boundto the IDE-beads but not control beads (FIG. 1). The IDE-beads wereunable to adsorb ³⁵S-labeled luciferase as the negative control,demonstrating the specificity of the observed binding.

Example 3 Genomic Structure of ALEX1 Gene

BLASTN analysis against GenBank and EMBL databases revealed that theclone U61B11 derived from the human chromosome X (GenBank Accession No.Z73913), sequenced by the Sanger Center, contains the sequence of theALEX1 gene. Therefore, the inventors used this genomic sequence todetermine the exon/intron organization of ALEX1 gene. As a result, itwas revealed that the ALEX1 gene comprises 4.2 kb in length and iscomposed of four exons, ranging in size from 54 to 1892 nucleotides,with the coding region residing entirely in a single exon (exon 4). Allexon-intron junctions conform to the consensus sequence for splicedonor/acceptor sites (gt/ag).

Example 4 Homology Search of Human ALEX1 Protein with Other Proteins

A homology search of public databases using the BLASTP program revealedthat the full-length ALEX1 protein shares highest homology to previouslyuncharacterized ORF KIAA0512 of 632 amino acids. The homology region ispresent in the C-terminal parts of the both amino acid sequences with51% identity and, when substitutions of similar amino acids were takeninto account, there was a 72% homology over the 234 amino acids region(FIGS. 2 to 3). ALEX1 and KIAA0512 proteins have also almost identicalN-terminal amino acid sequence (amino acid position 1 to 25) predictedto target these proteins to the endoplasmic reticulum (ER) membranethrough this region. Similarly with ALEX1, the C-terminus of KIAA0512contains microbodies targeting signal.

Although weaker than KIAA0512, ALEX1 shares homology to anotheruncharacterized ORF KIAA0443 (31% identity and, when substitutions ofsimilar amino acids were taken into account, there was 55% homology overthe 225 amino acids region) (FIGS. 2 to 3).

ALEX1 sequence was analyzed also by PSI-BLAST program. The PSI-BLASTmethod significantly increases the database-search sensitivity byincorporating information embedded in a multiple sequence alignment intoa position-dependent weight matrix, which is employed as the query foriterating the search (Altshul, S. F. et al. (1997) Nucleic Acids Res.25, 3389-3402). This allows the detection even of very low sequencehomologies at a statistically significant level. Second iteration withE-value of 0.01 actually revealed that ALEX1 exhibits significanthomologies to members of the Arm repeat family. In this search, ALEX1showed the highest homology to importin α and yeast vacuolar protein 8(Vac8p). Lower homology is shared with armadillo, β-catenin, andplakoglobin. The homology region is confined to the two Arm repeatregions present in ALEX1 protein.

BLASTN searches of dbEST database with the ALEX1 cDNA sequence as aquery identified a number of highly homologous ESTs. These containedESTs corresponding to the above-mentioned KIAA0512, KIAA0443 and anadditional previously unknown cDNA. When several representative ESTs forthis unknown cDNA were screened trough the dbTHC database at NCBI, thecontig sequence of 2,036 nucleotides composed of 44 ESTs was identified(THC257925). A conceptual translation product of the THC257925 is 342amino-acid protein with strong homology to ALEX1, KIAA0512 and theC-terminal half of KIAA0443 (FIGS. 2 to 3). This novel protein shares55% identity and, when substitutions of similar amino acids were takeninto account, there was 74% homology over the region of 259 amino acidswith ALEX1 protein. Pfam algorithm detects in this amino acid sequenceone Arm motif sequence, which corresponds to the first Arm motif inALEX1.

No protein with less than six Arm repeats has been described so faramong the known proteins of Arm family, and the classical members of thearm-family proteins including catenins and importins contain six or moreArm repeats. Thus, ALEX1 and its homologues do not represent truemembers of Arm repeat family proteins and constitute rather a newfamily, which the inventors name as ALEX family. Therefore, KIAA0512 andthe novel ORF THC257925 are named as “ALEX2” and “ALEX3”,correspondingly.

Example 5 Determination of Chromosomal Location of ALEX Genes

The search of the UniGene collection at NCBI established that the 3′region of ALEX1 corresponds to cluster Hs.9728. This cluster is linkedto the chromosomal marker sequence stSG9550 (DXS990-DXS1059). Similarly,the cDNA sequences of ALEX2 (KIAA0512) and ALEX3 (THC257925) match tothe clusters Hs.48924 and Hs. 172788, respectively, linked to stSG22124and stSG13135 that reside at the same interval DXS990-DXS1059. TheDXS990-DXS1059 interval is mapped to band q21.33-q22.2 on the long armof human chromosome X (Xq21.33-q22.2). BLASTN analysis further revealedthat a clone 769N13 from human chromosome Xq22.1-23 region sequenced atthe Sanger Center comprises the sequence of KIAA0443. Thus, alldescribed ALEX family genes including ALEX1 to 3 and KIAA0443 are shownto locate in the same region on human chromosome X.

Example 6 Analysis of ALEX1 and ALEX2 Gene Expression in Human Normaland Cancer Tissues

To investigate the gene expression of ALEX1 and ALEX2 in various humantissues, Northern blots prepared from poly (A)⁺ RNA purified from avariety of human tissues were hybridized with the probes derived fromthe region of least homology between the two ALEXs.

Human Multiple Tissue Northern Blots containing 2 μg each of poly(A)⁺RNA from various tissues and ExpressHyb hybridization solution werepurchased from Clontech. Hybridization was conducted with labeled probeat 1×10⁶ cpm/ml for 1 h at 68° C. The filters were washed with a finalstringency of 0.1×SSC, 0.1% SDS at 50° C. for 40 min and exposed toHyperfilm using intensifying screens at −80° C. The probes used weregenerated by PCR, purified through electrophoresis, and ³²P-labeled asdescribed above. The 227-bp ALEX1 probe was generated using primersF2/R2. The 328-bp ALEX2 probe was prepared with primers F4/R4.

As can be seen in FIG. 4, when ALEX1 probe was used, a clear single bandcorresponding to a transcript of about 2.2-kb was detected in themajority of the tissues. The size of the transcript indicates that thecloned cDNA represents the full-length ALEX1 mRNA. When the blots werehybridized with a probe specific for ALEX2, a transcript of about 2.7-kbwas detected in most tissues. An additional transcript of 1.4 kb isprominently present in the skeletal muscle, testis, and placenta, andweak band of about 7 kb is seen in the brain. The analysis reveals thatALEX1 and ALEX2 have remarkably similar distribution of expression. BothmRNA were higher in ovary, heart, testis, prostate, brain, spleen, andcolon. The transcripts were only barely detectable in liver and thymus.The expression of both mRNA in peripheral blood leukocytes was below thelimits of detection. Even highly sensitive RT-PCR failed to detect ALEX1and ALEX2 transcripts in leukocytes (data not shown).

Next, the expression of ALEX1 and ALEX2 in various human tumors wasanalyzed by RT-PCR. cDNA prepared from five normal tissues correspondingto respective tumors were used as positive controls. PCR analysis of theexpression of ALEX1 and ALEX2 in human normal and cancer tissues wasperformed with Human I, Human II, and Human Tumor Multiple Tissue cDNApanels from Clontech. Primer combinations were F3/R2 for ALEX1 and F5/R5for ALEX2. As shown in FIG. 5, no expression of ALEX1 and ALEX2 mRNA wasdetected in two lung carcinoma samples, in a prostatic adenocarcinomasample, and in a colon adenocarcinoma sample. In addition, ALEX2transcripts were not detectable in pancreas and ovarian carcinomasamples, while ALEX1 expression was significantly reduced in thesesamples as compared to the corresponding normal tissues. cDNA used inthese experiments were prepared from tumor tissues that have beenexplanted and propagated as xenografts in nude mice.

To eliminate the possibility of changes of cell characteristics duringxenograft maintenance, the inventors utilized the blot containing mRNAisolated from tumor ovaries and corresponding normal tissues of ovariesfrom four different donors to investigate the expression of ALEX1.Northern Territory Ovarian Tumor Blot containing 20 μg each of RNAisolated from human normal tissues and ovarian cancer tissues excised atthe same operational site was obtained from Invitrogen. Northernhybridization was performed as described above. As shown in FIG. 6,ALEX1 mRNA is expressed in the normal part but undetectable in the tumorpart of the ovaries from all four donors.

Example 7 Investigation of Expression Pattern of ALEX1 and ALEX2 inVarious Human Tumor-Derived Cell Lines

Using RT-PCR, the present inventors examined ALEX1 and ALEX2 expressionin various human tumor-derived cell lines. Namely, 2 μg of total RNA wasreverse-transcribed using the Superscript II first-strand cDNA synthesiskit (Gibco BRL) with oligo(dT) primers according to the manufacturer'sspecifications. Obtained cDNA was subjected to PCR amplification usingprimers described above. As shown in FIG. 7, four normal tissues and twonormal human diploid fibroblast cell lines were included as positivecontrols. Both transcripts are expressed in the glioblastoma-derivedcell line A172 and the osteosarcoma-derived cell line TE-85. Low levelsof ALEX1 and ALEX2 expression were found in the osteosarcoma-derivedcell line U-2OS. However, no signal was detected in the immortalendothelial cell line t-HUE2, the cervix adenocarcinoma cell line HeLa,the lung carcinoma cell line A549, the non-small cell lung carcinomacell line ABC-1, the cervical adenocarcinoma cell line C-33, andcytomegalovirus-transformed embryo lung fibroblast cell lineCMV-Mj-HEL-1. The present inventors also examined ALEX1 and ALEX2expression in four additional cell lines. These four cell lines were thenormal human mammary gland epithelial cell line HBL-100, the breastadenocarcinoma cell line MDA-MB-468, and the neuroblastoma cell linesSH-SY5Y and IMR-32. ALEX1 and ALEX2 mRNA was detected in HBL-100 andboth neuroblastoma cell lines but not in MDA-MB-468 (data not shown).Thus, it was shown that ALEX1 and ALEX2 are expressed in human sarcoma,glioblastoma, and neuroblastoma-derived cell lines but are notdetectable in cell lines derived from human carcinomas of epithelialorigin.

Example 8 ALEX1 is Developmentally Regulated

Next the present inventors analyzed expression of ALEX1 mRNA atdifferent stages of mouse embryo development. Based on the sequenceavailable from mouse EST database, the inventors designed a probe forisolating the mouse homologous gene of ALEX1. As seen in FIG. 8, mouseALEX1 transcript has a size of about 2.3-kb, practically identical tohuman ALEX1. Low levels of ALEX1 expression are already detectable in 7day-old mouse embryo and the levels markedly elevated between day 7 and11.

Example 9 Yeast Two-Hybrid Screen for ALEX1-Interacting Proteins

The yeast two-hybrid method was used to identify proteins that interactwith the ALEX1. Full-length ALEX1 was linked to Gal4-DB (DNA-bindingdomain of Gal4) in frame, and inserted into pODB-80 vector. Theresulting plasmid was used as a bait to perform two-hybrid screening asExample 1. As a result of screening human brain cDNA library usingfull-length ALEX1 cDNA as the bait, three independent clonescorresponding to overlapping fragments of JNK interacting protein-1(JIP-1) have been identified. These represented amino acid numbers 160to 711, 199 to 711, and 258 to 711 of human JIP-1. JIP-1 wascharacterized initially as a JNK-interacting protein and inhibitor ofJNK signaling, by virtue of its ability to prevent nuclear translocationof JNK (Dickens, M. et al. (1997) Science 277, 693-696). To identify thebinding site of ALEX1 on JIP-1, various fragments of JIP-1 were preparedand used in the yeast two-hybrid assay. As shown in FIG. 9, the resultsindicate that the region of amino acid numbers 374 to 479 of JIP-1 ismainly involved in ALEX1 binding. However, the residues N-terminal toamino acid number 374 and C-terminal to amino acid number 479 might benecessary for more rigid binding because a fragment comprising aminoacid numbers 262 to 589 conferred faster growth of yeast cells ascompared to that for amino acid numbers 374 to 711 (FIG. 9).

Example 10 Effect of ALEX1 Overexpression on c-Jun-DependentTranscriptional Activation

Because ALEX1 binds to JIP-1 in the yeast two-hybrid screen, the effectsof forced expression of ALEX1 in cells lacking ALEX1 expression on JNKsignaling pathway were investigated. Activated JNK phosphorylatestranscriptional factor c-Jun whereby increasing its transcriptionactivating ability. Therefore, for the experiments the human cervicalcarcinoma cell line HeLa S3 which does not express ALEX1 was used.

Gal4-c-Jun fusion protein was used as c-Jun, and its transcriptionactivating ability was measured in co-transfection assays with thereporter plasmid pG5-Luc. This pG5-Luc contains five GAL4 sites clonedupstream of a minimal promoter element and the firefly luciferase gene.The plasmid pRL-TK having Renilla luciferase gene under the control ofthymidine kinase gene promoter was co-transfected to provide an internalcontrol. These plasmids were transfected into HeLa cells with GenePORTERtransfection reagent (Gene Therapy Systems) in 12-well dishes. Aftertransfection, cells were cultured in 0.5% FBS and reporter activitieswere analyzed after 42 h.

The MEKK1 protein, a known JNK activator, increased the expression ofthe c-Jun-dependent luciferase reporter gene by about 30-fold (FIG. 10),indicating activation of the Gal4-cJun fusion protein. The pFC-dbdplasmid (Gal4), which comprises the Gal4 DNA binding domain only and noactivation domain, could not be activated by overexpression of the MEKK1demonstrating the specificity of the assay. Forced expression of theALEX1 significantly inhibited MEKK1-induced c-Jun-dependenttranscription activating ability with the inhibition displaying adose-dependent manner (FIG. 10).

Example 11 Interaction Between ALEX1 and Presenilin-1 (PS1)

Interaction between ALEX1 and presenilin-1 (PS1) was investigated bypull-down assay. The large loop region of PS1 protruding into thecytoplasm reported to be the binding site with delta-catenin (PS1-CL:amino acid numbers 263-407 of PS-1 according to accession numberL76517), and the N-terminal region (PS1-N: amino acid numbers 1-81 ofPS-1 according to accession number L76517) as a negative controlrespectively were cloned into pGEX-5X-1 by PCR method, and wereexpressed in E. coli as GST fusion proteins. GST or GST fusion proteins,GST-PS1-CL (263^(rd) to 407^(th) amino acids) and GST-PS1-N (1^(st) to81^(st) amino acids), were immobilized onto glutathione-Sepharose beadsby incubating 80 μg of the proteins with 20 μl of the beads for 16 hr at4° C. in immobilization buffer (20 mM Tris/Cl, pH 7.2 at 25° C., 140 mMNaCl, 1 mM EDTA, 0.1% Tween-20 and Complete™ Protease Inhibitor,Boehringer Mannheim). The mixture was centrifuged, unbound protein wasdiscarded, and the beads were resuspended in 500 μl of binding buffer(20 mM Tris/Cl, pH 7.2 at 25° C., 140 mM NaCl, 0.1% Tween-20, 0.1 mMDTT, 4 mg/ml BSA and Complete™ Protease Inhibitor). In vitro-translated,[³⁵S]methionine-labeled (Promega TNT reticulocyte lysate system) ALEX1was added to the mixture and incubated for 2.5 h at 4° C. to formcomplexes. Instead of ALEX1, luciferase as a negative control, anddelta-catenin (615-1225) and delta-catenin (826-1225) as positivecontrols were used for same experiments. The beads were washed fivetimes in wash buffer (20 mM Tris/Cl, pH 7.2 at 25° C., 140 mM NaCl, 0.1%Tween-20 and Complete™ Protease Inhibitor), resuspended in 40 μl of2×SSC sample buffer and incubated at 90° C. A half of the eluted proteinwas resolved by SDS-PAGE (4-20%) and visualized by BAS2000.

As a result, bands were confirmed by coprecipitation of the labeledprotein by GST-PS1-CL, only in 2 types of delta-catenin fragments and inALEX1. These bands were not confirmed by GST or GST-PS1-N. Also for thenegative control luciferase, bands were not confirmed at all.

Example 12 Search of ALEX1 Binding Proteins by the Yeast Two-HybridMethod

Since many false positive clones were obtained as a result ofpreliminary examination of yeast two-hybrid method using full-lengthALEX1 as a bait, further examinations were made. It was elucidated thata large reduction in the number of false positive clones is possible byusing as a bait, ALEX1 in which the transmembrane domain (amino acidnumbers 1 to 27) existing at the N-terminal is removed. Therefore, aftertransforming yeast PJ69-2A with an expression vector in which delta(1-27) ALEX1 is linked to Gal4-DBD, human brain-derived cDNA librarythat is cloned onto a pACT2 vector (Human Brain Matchmaker cDNA Library:Clontech) was directly transfected, and then selection of nutritionaldemand indicated below was performed. As a result of screening a totalof 2,100,000 clones, 476 clones grew under -His conditions, andfurthermore approximately 40% of these were able to grow under -His/-Adeconditions. Further selection was continued, and ultimately 180 cloneswere selected under -His/-Ade/-Leu/-Trp conditions. Next, by rescuinglibrary plasmids from these positive colonies to E. coli, and collectingthem upon purification, nucleotide sequence determination on a total of155 clones were performed. As a result, it was elucidated thatapproximately 10% of the colonies simultaneously carried two differentlibrary plasmids, and in such cases, the possibility that one of themwas a false positive was considered. Therefore, for the purpose offurther eliminating false positive clones, co-transfection withGal4-DBD-delta (1-27) ALEX1 or Gal4-DBD that does not contain the baitinto yeast PJ69-2A was further performed on all 155 clones obtained, andthen clones capable of bait-dependent growth under -His/-Leu/-Trpconditions were selected. From this assay, approximately 20% of theclones could be eliminated as being false positive. Next, aftertransforming yeast Y187 strain with the same combination of plasmids,β-gal assay was performed. Several colonies showed negative β-galactivity. The following lists those that passed all of theabove-mentioned tests, and in addition that were included in two or moreindependent clones, and summarizes their expected functions.

p0071 (plakophilin-4): Arm repeat-containing presenilin binding protein

-   -   GenBank Ac. No. X81889; H. sapiens mRNA for p0071 protein,        GenBank Ac. No. NM_(—)003628; Homo sapiens plakophilin 4 (PKP4),        mRNA        SART-1: human squamous cell carcinoma antigen, expressed in        growing cells only    -   GenBank Ac. No. AB006198; Homo sapiens mRNA for SART-1, GenBank        Ac. No. Y14314; Homo sapiens mRNA for IgE autoantigen        MSP58: nucleoprotein, interacting to the growth-related        nucleoprotein p120, expressed in growing cells only    -   GenBank Ac. No. AF015308; Homo sapiens nucleolar protein (MSP58)        mRNA, GenBank Ac. No. AF068007; Homo sapiens cell        cycle-regulated factor p78 mRNA        ATRX: murine colon adenocarcinoma antigen, cell        cycle-dependently phosphorylated, a helicase/ATPase member of        the SNF2 family    -   GenBank Ac. No. U72938; Homo sapiens putative DNA dependent        ATPase and helicase (ATRX) mRNA, alternatively spliced product        3, GenBank Ac. No. NM_(—)000489; Homo sapiens alpha        thalassemia/mental retardation syndrome X-linked (RAD54 (S.        cerevisiae) homolog) (ATRX), mRNA        CSA2 (RED protein): chondrosarcoma-associated protein,        distributed in nuclei as dots, transcription-related function?    -   GenBank Ac. No. AF182645; Homo sapiens chondrosarcoma-associated        protein 2 (CSA2) mRNA        p68: RNA helicase/ATPase    -   GenBank Ac. No. X52104; Human mRNA for p68 protein, GenBank Ac.        No. NM_(—)004396; Homo sapiens DEAD/H (Asp-Glu-Ala-Asp/His) box        polypeptide 5 (RNA helicase, 68 kD) (DDX5), mRNA        OS-9: amplified in human sarcoma, containing nuclear        localization signal    -   GenBank Ac. No. NM_(—)006812; Homo sapiens amplified in        osteosarcoma (OS-9), mRNA        ZNF189: C₂H₂ Zinc Finger protein    -   GenBank Ac. No. AF025770; Homo sapiens C2H2 zinc finger protein        (ZNF189) mRNA        KIAA1221: C₂H₂ Zinc Finger protein    -   GenBank Ac. No. AB033047; Homo sapiens mRNA for KIAA1221        protein, partial cds        α-Actinin4: involved in actin polymerization, cell motility, and        cancer infiltration    -   GenBank Ac. No. NM_(—)004924; Homo sapiens actinin, alpha 4        (ACTN4) mRNA        ZIP kinase    -   GenBank Ac. No. AB022341; Homo sapiens mRNA for ZIP kinase        ALEX1: homodimer formation

Example 13 Examination of Intracellular (COS7 Cell) Localization UsingALEX1 with GFP Added to the N or C-Terminal End

When a fusion protein in which ALEX1 was added to the C-terminal(N-EGFP-ALEX1-C) or to the N-terminal (N-ALEX1-EGFP-C) of EGFP wasexpressed in COS7 cells, and the intracellular localization was examinedusing a fluorescence microscope, it was confirmed that N-EGFP-ALEX1-Cwas distributed as spots within the nucleus (thought to be nucleoli),and in contrast, N-ALEX1-EGFP-C was observed to be eliminated from thenucleus and localized in the cytoplasm. This result was thought to bedue to masking of the transmembrane domain existing near the N-terminalof ALEX1 in N-EGFP-ALEX1-C by added GFP. Therefore, the possibility ofregulation of intracellular localization mediated by blocking orenzymatic cleavage of the transmembrane domain of ALEX1 was suggested.

INDUSTRIAL APPLICABILITY

This invention provides a novel armadillo repeat-containing proteinALEX1, and its gene. Expression of ALEX1 protein is significantlydecreased in epithelial cancer. ALEX1 is also expected to be involved inAlzheimer's disease. Accordingly, by detecting the expression level ofALEX1, it is possible to test for cancer and Alzheimer's disease. Inaddition, it is possible to develop new pharmaceutical agents forpreventing or treating these diseases by screening for drugs thatregulate the expression or activity of ALEX1.

1. An isolated nucleic acid molecule comprising: (a) a polynucleotideencoding a protein, which comprises the amino acid sequence of the humanALEX1 protein of SEQ ID NO: 3; or (b) a nucleic acid sequence fullycomplementary to (a).
 2. The isolated nucleic acid molecule of claim 1,wherein the polynucleotide comprises the nucleic acid sequence of SEQ IDNO: 1 or SEQ ID NO:
 2. 3. A vector comprising the isolated nucleic acidmolecule of claim
 1. 4. An isolated host cell comprising the nucleicacid molecule of claim 1 or the vector of claim
 3. 5. A method ofproducing a recombinant protein, the method comprising: (a) cultivatingthe host cell of claim 4; and (b) collecting or isolating the expressedrecombinant protein from the host cell or culture supernatant thereof.6. A kit comprising a testing reagent for cancer or Alzheimer's disease,comprising a probe or primer comprising the isolated nucleic acidmolecule of claim
 1. 7. A probe or primer comprising the isolatednucleic acid molecule of claim 1 and a label.
 8. The probe or primer ofclaim 7, wherein the label comprises ³²P and the nucleic acid moleculecomprises a PCR generated ³²P-labeled probe or primer.
 9. A kitcomprising the probe or primer of claim 7.